Compositions and methods for the treatment of cancer

ABSTRACT

Disclosed herein are methods and compositions for the treatment of cancer. In particular, the present invention discloses inhibitors of the Fanconi anemia pathway and methods using same. Such inhibitors are useful in inhibiting DNA damage repair and can be useful, for example, in the treatment of cancer.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application60/684,136, filed May 24, 2005. This application is also acontinuation-in-part of co-pending U.S. application Ser. No. 10/165,099,filed Jun. 6, 2002, which is a continuation-in-part of U.S. applicationSer. No. 09/998,027, filed Nov. 2, 2001, now abandoned, which claims thebenefit of U.S. provisional application 60/545,756, filed Nov. 3, 2000.This application is also a continuation-in-part of co-pending U.S.application Ser. No. 11/046,346, filed Jan. 28, 2005, which claims thebenefit of U.S. provisional application 60/540,380, filed Jan. 30, 2004.Each of the aforementioned applications is hereby incorporated byreference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grants DK43889 andHL52725 from the National Institutes of Health. The U.S. Government hascertain rights in the invention.

FIELD OF THE INVENTION

This invention generally relates to compositions and methods for thetreatment of cancer.

BACKGROUND OF THE INVENTION

Many kinds of cancer resist effective chemotherapeutic treatment. Inovarian cancer, resistance is observed towards chemotherapeutic agentssuch as cisplatin. Cisplatin (cis-diamminedichloroplatinum, or CDDP),discovered originally in the late 1960s, is a cytotoxic drug used totreat many cancers, including ovarian cancer. Cisplatin acts byplatination of DNA, resulting in DNA crosslinking. Up to 50% of ovariancarcinomas are intrinsically resistant to conventional chemotherapeuticagents such as cisplatin or other related platinum therapies. Manymechanisms of resistance have been postulated. However, the precisemechanism(s) underlying the intrinsic and extrinsic resistance tochemotherapy has not been elucidated. One method of reversing resistanceto chemotherapy involves the use of chemosensitizers. Chemosensitizersgenerally inhibit the mechanism of resistance. Examples includeverapamil, reserpine, tamoxifen and cremophor, inhibitors of effluxpumps conferring multidrug resistance (MDR1, P-glycoprotein). However,such chemosensitizers are effective only in a subset of tumors wheredrug efflux is the main mechanism of resistance. In addition, a numberof these chemosensitizers have undesirable side effects.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of predicting whether asubject with a neoplastic disorder or disease will respond to agenotoxic anti-neoplastic agent. The method comprises obtaining abiological sample from the subject, and determining degree ofubiquitination of the Fanconi anemia complementation group D2 (FANC D2)polypeptide within the biological sample. A degree of ubiquitination ofthe FANC D2 polypeptide in the biological sample of the subject that isless than about 70% when compared with a biological sample from acontrol subject is indicative of a subject that will respond to agenotoxic anti-neoplastic agent.

In another aspect, the invention provides a method of predicting whethera subject with a neoplastic disorder or disease will respond to agenotoxic anti-neoplastic agent. The method comprises obtaining abiological sample from the subject, and determining the FANCD2-containing foci within the biological sample. A difference in fociformation, wherein the sample from the subject that contains less thanabout 70% of the FANC D2-containing foci when compared with thebiological sample from a control subject is indicative of a subject thatwill respond to a genotoxic anti-neoplastic agent.

In another aspect, a method of identifying an inhibitor of a non-FA DNAdamage repair pathway is provided. The method comprises the followingsteps: (a) providing a control cell that is functional in the FanconiAnemia (FA) pathway; (b) providing a test cell that is isogenic to thetest cell but is defective in the FA pathway; (c) contacting the testcell and the control cell with a test compound; and, (d) comparing thesensitivity of the test cell and said control cell to the test compound.An increased sensitivity of the test cell to the test compound than thecontrol cell is indicative of a test compound being an inhibitor of anon-FA DNA damage repair pathway.

In yet another aspect, a method of treating a neoplastic disorder in asubject in need thereof is provided. The method comprises administeringto the subject a combination of an effective amount of: (a) an inhibitorof the FA pathway or pharmaceutically acceptable salts, esters,derivatives, solvates or prodrugs thereof, and (b) a genotoxicanti-neoplastic agent.

In still another aspect, a method of treating a neoplastic disorder in asubject in need thereof is provided. The method comprises administeringto the subject a combination of an effective amount of: (a) an inhibitorof the FA pathway or pharmaceutically acceptable salts, esters,derivatives, solvates or prodrugs thereof, and (b) an inhibitor of anon-FA DNA damage repair pathway.

In another aspect, a method of treating a neoplastic disorder in asubject in need thereof is provided. The method comprises administeringto the subject a combination of an effective amount of: (a) an inhibitorof the FA pathway or pharmaceutically acceptable salts, esters,derivatives, solvates or prodrugs thereof, (b) an inhibitor of a non-FADNA damage repair pathway, and (c)a genotoxic anti-neoplastic agent orpharmaceutically acceptable salts, esters, derivatives, solvates orprodrugs thereof.

In another aspect, a method of increasing the sensitivity of aneoplastic disorder to a genotoxic anti-neoplastic agent is provided.The method comprises administering, before, after or concurrently with atherapeutically effective dose of the agent an effective amount of aninhibitor of the FA pathway.

In a final aspect, a method of increasing the sensitivity of aneoplastic disorder to a genotoxic anti-neoplastic agent is provided.The method comprises administering, before, after or concurrently with atherapeutically effective dose of the agent a combination of aneffective amount of (a) an inhibitor of the FA pathway orpharmaceutically acceptable salts, esters, derivatives, solvates orprodrugs thereof, and (b) an inhibitor of a non-FA DNA damage repairpathway.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 outlines an example of the workflow of screens for theidentification of inhibitors and agonists of the FA pathway.

FIG. 2 outlines the high-throughput scheme for identification of FApathway agonists and antagonists using fluorescence microscopy.

FIG. 3 is a schematic showing the protein components identified withinthe FA pathway of DNA damage repair

FIG. 4 shows the schematic of eGFP-FANC D2 fusion construct, and itsubiquitination in transfected PD20F, GM6914 and HeLa cells upon exposureto ionizing radiation (IR), HU, or Mitomycin C (MMC).

FIG. 5 are fluorescence micrographs showing the fluorescence signalemitted by eGFP-FANC D2 transfected PD20 and GM6914 cells. GM6914 cellsadditionally transfected with FANCA shows punctate, FANC D2-containingfoci upon exposure to ionizing radiation.

FIG. 6 outlines a method for screening for FA inhibitors by fluorescencemicroscopy.

FIG. 7 are fluorescence micrographs showing inhibition of IR-mediatedformation of FANC D2-containing foci upon exposure of eGFP-FANC D2containing cells to the kinase inhibitor H-9.

FIG. 8 shows an immunoblot analysis showing the effect of H-9 oninhibition of IR-inducible monoubiquitination of FANC D2 (top twopanels), phosphorylation of ATR kinase (middle panel), Chk1phosphorylation (fourth panel), and Chk1 polypeptide levels (bottompanel). The graph shows the enhanced sensitivity of 2008 cells tocisplatin by H-9. The 2008 cells are inherently sensitive to cisplatinbecause of a deficiency FANCF (Open Square). This sensitivity tocisplatin is reduced upon transfection of FANCF (2008+F, open circle).Treatment of 2008+F cells with H-9 re-establishes sensitivity tocisplatin (closed circle), while not affecting sensitivity to cisplatinin untranfected cells (closed square).

FIG. 9 shows the inhibition of cisplatin-dependent FANC D2monoubiquitination by treatment with curcumin (9A). The graph (9B) showsthe sensitization of FANCF-corrected 2008 cells to cisplatin, but areduced sensitizing effect on the chemosensitization of the parental2008 cells by curcumin treatment.

FIG. 10 shows the effects of curcumin on FANCD2 foci, and sensitivitytowards cisplatin. (a) Curcumin sensitizes human tumor cells toCisplatin. In this experiment we compared 2008 cells (an ovarian tumorline which is deficient in the FANCF protein and therefore has a defectin the FA pathway) and 2008 cells corrected with the FANCF cDNA. Thecells were pretreated for 24 hours with or without curcumin (20micromolar) as indicated, and the cells were then exposed to increasingdoses of cisplatin. Importantly, the corrected 2008 cells are sensitizedto cisplatin by pretreatment with curcumin. (b) Pretreatment of Clone 7cells with Curcumin prevents the assembly of FANCD2 foci.

FIG. 11 shows an immunoblot analysis showing the effect ofalsterpaullone on inhibition of IR-inducible monoubiquitination of FANCD2. Alsterpaullone, a Cdk1 inhibitor, does not inhibit IR-inducible D2phosphorylation on T691 but rather enhances it. Alsterpaullone inhibitsthe IR-inducible monoubiquitination of FANC D2 and phosphorylation ofChk1 (on Ser345) in an ovarian cancer cell line.

FIG. 12 are micrographs showing inhibition of IR-mediated FANC D2- andBRCA1-containing foci in cells treated with alsterpaullone.

FIG. 13 shows that treatment of cells with spermine NONOate inducesphosphorylation and monoubiquitination of FANC D2, and phosphorylationof Chk1 in the absence of IR.

The graph shows a nominal reduction in sensitivity of 2008 cells tospermine NONOate upon FANC F transfection (2008+F), when compared withvector transfected 2008 cells (2008+vec).

FIG. 14 shows that treatment of cells with spermine NONOate inducesphosphorylation and monoubiquitination of FANC D2, and phosphorylationof Chk1 in the absence of IR.

FIG. 15 are micrographs showing that spermine NONOate inducesphosphorylation of histone H2AX in the absence and in the presence of IRtreatment.

FIG. 16 shows an immunoblot analysis showing that geldanamycin (an Hsp90inhibitor) inhibits IR-inducible D2 monoubiquitination in HeLa cells.Geldanamycin also causes decreased Chk1 expression. The graph shows thattransfection of 2008 cells with FANC F does not alter sensitivity ofcells to geldanamycin.

FIG. 17 are micrographs showing inhibition of IR-mediated FANC D2- andBRCA1-containing foci in cells treated with geldanamycin.

FIG. 18 shows the effects of Go6976, a PKC, Chk1 inhibitor, onIR-inducible monoubiquitination of FANC D2 in HeLa cells. Go6976enhances phosphorylation of Chk1 in HeLa cells. However, transfection of2008 cells with FANC F has no effect in sensitivity to Go6976.

FIG. 19 are micrographs showing inhibition of IR-mediated FANCD2-containing foci formation in cells treated with Go6976.

FIG. 20 are immunoblot panels showing the effects of AG370, a PDGFRkinase inhibitor, in inhibiting IR-inducible monoubiquitination of FANCD2 in HeLa cells. The effects of AG370 on Chk1 is not clear.

FIG. 21 are micrographs showing inhibition of IR-mediated FANC D2- andBRCA1-containing foci in cells treated with AG370.

FIG. 22 shows that inactivation of the FA pathway causes increasedsensitivity to DNA cross-linking agents, including BCNU. (a) IsogenicPD20 (circle) and PD20 retrovirally corrected with FANCD2 (square)showed differential sensitivity to BCNU. Percentage survival was plottedon the Y-axis. Similar data were obtained using 2008 (FANCF deficientcell line) and 2008 complemented with FANF. (b) Model of FA pathway. (c)BCNU induced FANCD2 monoubiquitination only in the presence of an intactFA pathway (lane 4). 2008 and 2008 retrovirally complemented with FANCFwere either untreated or treated with BCNU. Whole cell lysates werefractionated on SDS-PAGE and immunoblotted with FANCD2 antisera. Similardata were derived using PD20 and PD20 corrected with FANCD2. (d) HT15GBM cell lines failed to undergo FANCD2 monoubiquitination upon BCNUtreatment. Other GBM lines tested (LN308, LN428, A172, T98G as shownabove; CRL1620, CRL2020, U87, U343 (not shown)) underwent appropriateFANCD2 monoubiquitination on treatment with BCNU. (e) Failure to undergoFANCD2 monoubiquitination in HT15 cell lines correlated with increasedsensitivity to BCNU. Percentage survival was plotted on the Y-axis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on a series of discoveriesshowing the critical role played by the FA pathway in the sensitivity ofcancers to anti-neoplastic agents. The role of the FA pathway inmodulating the sensitivity of neoplastic disorders and/or cancer cellsto anti-neoplastic agents has been demonstrated using cell linesdeficient in FA pathway components, and using inhibitors of the FApathway. Therefore; in one embodiment, a method for treating a subjectwith a neoplastic disorder is provided. One such method comprisesadministering an effective dose of an FA pathway inhibitor incombination with a genotoxic anti-neoplastic agent. Another methodcomprises administering an effective dose of an FA pathway inhibitor incombination with an inhibitor of a non-FA DNA damage repair pathway.Also provided are compositions useful for the treatment of neoplasticdisorders comprising an FA pathway inhibitor in combination with agenotoxic anti-neoplastic agent and/or an inhibitor of a non-FA DNAdamage repair pathway. Also provided are pharmaceutical compositions, aswell as kits thereof, comprising FA pathway inhibitors andanti-neoplastic agents and/or an inhibitor of a non-FA DNA damage repairpathway.

Also provided are methods of identifying agents which modulate the FApathway. Such methods are useful in identifying inhibitors of the FApathway. Inhibitors thus identified are potentially useful aschemosensitizing and/or radiosensitizing agents. Also provided in thepresent invention are methods for identifying a non-FA DNA damage repairpathway inhibitor to be used in combination with the FA pathwayinhibitor. The combination of the inhibitors may be useful to administerto patients receiving anti-neoplastic agents.

I. Definitions

As used herein, the terms “neoplasm”, “neoplastic disorder”, “neoplasia”“cancer,” “tumor” and “proliferative disorder” refer to cells having thecapacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth which generally formsa distinct mass that show partial or total lack of structuralorganization and functional coordination with normal tissue. The termsare meant to encompass hematopoietic neoplasms (e.g. lymphomas orleukemias) as well as solid neoplasms (e.g. sarcomas or carcinomas),including all types of pre-cancerous and cancerous growths, or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. Hematopoietic neoplasms are malignant tumors affectinghematopoietic structures (structures pertaining to the formation ofblood cells) and components of the immune system, including leukemias(related to leukocytes (white blood cells) and their precursors in theblood and bone marrow) arising from myeloid, lymphoid or erythroidlineages, and lymphomas (relates to lymphocytes). Solid neoplasmsinclude sarcomas, which are malignant neoplasms that originate fromconnective tissues such as muscle, cartilage, blood vessels, fibroustissue, fat or bone. Solid neoplasms also include carcinomas, which aremalignant neoplasms arising from epithelial structures (includingexternal epithelia (e.g., skin and linings of the gastrointestinaltract, lungs, and cervix), and internal epithelia that line variousglands (e.g., breast, pancreas, thyroid). Examples of neoplasms that areparticularly susceptible to treatment by the methods of the inventioninclude leukemia, and hepatocellular cancers, sarcoma, vascularendothelial cancers, breast cancers, central nervous system cancers(e.g. astrocytoma, gliosarcoma, neuroblastoma, oligodendroglioma andglioblastoma), prostate cancers, lung and bronchus cancers, larynxcancers, esophagus cancers, colon cancers, colorectal cancers,gastro-intestinal cancers, melanomas, ovarian and endometrial cancer,renal and bladder cancer, liver cancer, endocrine cancer (e.g. thyroid),and pancreatic cancer.

A “genotoxic agent” or “genotoxin” refers to any chemical compound ortreatment method that induces DNA damage when applied to a cell. Suchagents can be chemical or radioactive. A genotoxic agent is one forwhich a primary biological activity of the chemical (or a metabolite) isalteration of the information encoded in the DNA. Genotoxic agents canvary in their mechanism of action, and can include: alkylating agentssuch as ethylmethane sulfonate (EMS), nitrosoguanine and vinyl chloride;bulky addition products such as benzo(a)pyrene and aflatoxin B1;reactive oxygen species such as superoxide, hydroxyl radical; baseanalogs such as 5-bromouracil; intercalating agents such as acridineorange and ethidium bromide.

A “genotoxic anti-neoplastic agent”, as used herein, is a genotoxicagent used for chemotherapy, for example, to treat cancer. Inparticular, “genotoxic anti-neoplastic agents” encompass agents, bothchemical or otherwise, which cause damage to DNA. These agents includeDNA alkylating agents, intercalating agents, and the like. Non-limitingexamples of “genotoxic anti-neoplastic agents” include1,3-Bis(2-Chloroethyl)-1-NitrosoUrea (BCNU), Busulfan, Carboplatin,Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine,Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Idarubicin,Ifosfamide, Irinotecan, Lomustine, Mechlorethamine, Melphalan, MitomycinC, Mitoxantrone, Oxaliplatin, Temozolomide, and Topotecan. “Genotoxicanti-neoplastic agents” also include radiation, in particular the typesused in radiation therapy for the treatment of cancer, in a dosagessufficient to cause damage to DNA in a subject.

“DNA damage”, as used herein, refers to chemical and/or physicalmodification of the DNA in a cell, including methylation, alkylationdouble-stranded breaks, cross-linking, thymidine dimers caused byultraviolet light, and oxidative lesions formed by oxygen radicalbinding to DNA bases.

As used herein, a “chemosensitizer” and “chemosensitizing agent” referto a compound which, when administered in a therapeutically effectiveamount in a subject, increases the sensitivity to chemotherapycompounds, and/or increases the therapeutic efficacy of the compounds,for example, in the treatment of a disease, such as neoplastic diseases,benign and malignant tumors, and cancerous cells. An increase insensitivity to chemotherapy compounds, including genotoxicanti-neoplastic agents, can be measured, for example, by measuring thedecrease in LD₅₀ of a cell towards a compound in the presence of thechemosensitizer.

Similarly, a “radiosensitizer” and “radiosensitizing agent”, as usedherein, refer to a compound which, when administered in atherapeutically effective amount in a subject, increases the sensitivityto radiation therapy (treatment by electromagnetic radiation), and/orincreases the therapeutic efficacy of radiation therapy, for example, inthe treatment of a disease, such as neoplastic diseases, benign andmalignant tumors and cancer cells. Also contemplated are electromagneticradiation treatment of other diseases not listed herein.

By “sample” or “biological sample” is meant any cell or tissue, or cellor tissue-containing composition or isolate derived from the subject.The sample may be derived from heart, brain, placenta, liver, skeletalmuscle, kidney, pancreas, spleen, thymus, prostate, testis, uterus,small intestine, or colon. Another type of biological sample may be apreparation containing white blood cells, e.g., peripheral blood,sputum, saliva, urine, etc., for use in detecting the presence orabsence of DNA damage in a subject that has been exposed to a genotoxicagent, such as radiation, chemicals, etc.

As used herein, “degree of ubiquitination” of the FANC D2 polypeptiderefers generally to the level of activation of the FA pathway, asmeasured by the degree of monoubiquitination of the FANC D2 polypeptidewithin a subject or biological sample therefrom. As used herein, the“degree of ubiquitination” of the FANC D2 polypeptide can encompass theproportion of total FANC D2 polypeptide within a sample that ismonoubiquitinated, and can be expressed on a fractional or percentagebasis. As used herein, the “degree of ubiquitination” of the FANC D2polypeptide can also be measured using any substitute methods ofdetecting activation of the FA pathway, including the degree of fociformation.

As used herein, “degree of foci formation” refers to the total number orthe rate of formation of FANC D2-containing foci in a sample. FANCD2-containing foci are nuclear protein complexes formed in response tothe activation of the FA pathway, for example by exposure to a genotoxicagent. FANC D2-containing foci can be detected, for example, byimmunofluorescence microscopy using a labeled antibody directed againstthe FANC D2 polypeptide, as further described herein. In certain cases,FANC D2-containing foci can also be detected in cells expressing afunctional fusion protein comprising GFP and the FANC D2 polypeptide. Inthese cells, FANC D2-containing foci can be detected using fluorescencemicroscopy without the use of anti-FANC D2 antibodies. The degree offoci formation can be normalized from one sample to another, forexample, to total number of cells, total number of intact nuclei, totalsample volume, or total sample mass.

By “difference in foci formation” is meant a difference, whether higheror lower, in the number, size or persistence of FANC D2-containing foci,when comparing a test sample with either a control sample or referencesample. A difference includes an increase or decrease that is 2-fold ormore, or less, for example 5, 10, 20, 100, 1000-fold or more as comparedto a control or reference sample. A difference also includes an increaseor decrease that is 5% more or less, for example, 10%, 20%, 30%, 50%,75%, 100%, as compared to a control or reference sample.

“Modulate” formation of FANC D2-containing foci, as used herein, refersto a change or an alteration in the formation of FANC D2-containing fociin a biological sample. Modulation may be an increase or a decrease infoci number, size or persistence within a biological sample, andincludes an increase or decrease that is 2-fold or more, or less, forexample 5, 10, 20, 100, 1000-fold or more as compared to a control orreference sample. Modulation may also be an increase or decrease that is5% more or less, for example, 10%, 20%, 30%, 50%, 75%, 100%, as comparedto a control or reference sample.

As used herein, exposure to a “low level” of a genotoxic anti-neoplasticagent refers to exposure to a dose of a particular genotoxicanti-neoplastic agent which results in no more than 20% of the maximalnumber of FANC D2-containing foci in biological samples. Because of themultitude of genotoxic anti-neoplastic agents to which a sample may beexposed, as well as the varying sensitivities of different samples tosuch genotoxic anti-neoplastic agents, it is preferable to express thedosage relative to the formation of FANC D2-containing foci, rather thanin the absolute dose of a particular genotoxic anti-neoplastic agent.

The term “modulator” refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian) cells or tissues, or even an inorganicelement or molecule. Modulators are evaluated for potential activity asinhibitors or activators (directly or indirectly) of a biologicalprocess or processes (e.g., agonist, partial antagonist, partialagonist, antagonist, anti-neoplastic agents, cytotoxic agents,inhibitors of neoplastic transformation or cell proliferation, cellproliferation-promoting agents, and the like) by inclusion in screeningassays described herein. The activities (or activity) of a modulator maybe known, unknown or partially-known. Such modulators can be screenedusing the methods described herein.

The term “candidate modulator” refers to a compound to be tested by oneor more screening method(s) of the invention as a putative modulator.Usually, various predetermined concentrations are used for screeningsuch as 0.01 μM, 0.1 μM, 1.0 μM, and 10.0 μM, as described more fullybelow. Test compound controls can include the measurement of a signal inthe absence of the test compound or comparison to a compound known tomodulate the target.

As used herein, an “FA pathway inhibitor” and “inhibitor of the FApathway” refer to a chemical compound (naturally occurring ornon-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian) cells or tissues, or even an inorganicelement or molecule. An “FA pathway inhibitor” and “inhibitor of the FApathway” refer broadly to compounds which inhibit the ability of the FApathway to repair DNA damage. Inhibition of the FA pathway by an “FApathway inhibitor” or an “inhibitor of the FA pathway” can be assessedusing the techniques described herein, including without limitation, thedetection of FANC D2-containing foci and detection of monoubiquitinationof the FANC D2 polypeptides. As will be appreciated by one skilled inthe art, the method contemplates any other method currently known orknown in the future, for the detection of the inhibition of the FApathway. Inhibition may be a decrease in number, size or persistence ofFANC D2-containing foci, and includes a decrease that is 2-fold or more,for example, 2, 5, 10, 20, 100, 1000-fold or more as compared to acontrol or reference. Inhibition may also be an decrease of 5% or more,for example 5%, 10%, 20%, 30%, 50%, 75%, or up to 100%, as compared to acontrol or reference. In addition, as used herein, an “FA pathwayinhibitor” and “inhibitor of the FA pathway” encompass thepharmaceutically acceptable salts, solvates, esters, derivatives orprodrugs.

A “non-FA DNA damage repair pathway”, as used herein, refers to any ofthe DNA damage repair pathways selected from the group consisting of thedirect reversal, non-homologous end joining (NHEJ), base excision repair(BER), nucleotide excision repair (NER), and mismatch repair (MR)pathways.

The pharmaceutical compositions of the present invention can beadministered using any amount and any route of administration effectivefor increasing the therapeutic efficacy of drugs. As used herein,“therapeutically effective amount,” when used in combination with achemosensitizer or radiosensitizer, refers to a sufficient amount of thechemosensitizing agent to provide the desired effect against targetcells or tissues. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject; the particular chemosensitizing agent; its mode ofadministration; and the like.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “therapeutically effective dose” refers to that amountof protein or its antibodies, antagonists, or inhibitors which preventor ameliorate the symptoms or conditions, for example, a neoplasticdisorder. Therapeutic efficacy and toxicity of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED₅₀ (the dose therapeutically effective in50% of the population) and LD₅₀ (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animalsstudies is used in formulating a range of dosage for human use. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage varies within this range depending upon the dosage from employed,sensitivity of the patient, and the route of administration.

The exact dosage is chosen by the individual physician or veterinarianin view of the patient to be treated. Dosage and administration areadjusted to provide sufficient levels of the active moiety or tomaintain the desired effect. Additional factors which may be taken intoaccount include the severity of the disease state; age, weight andgender of the subject; diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending on ahalf-life and clearance rate of the particular formulation.

The term “pharmaceutically acceptable salt” refers to both acid additionsalts and base addition salts. The nature of the salt is not critical,provided that it is pharmaceutically acceptable. Exemplary acid additionsalts include, without limitation, hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric, phosphoric, formic, acetic,citric, tartaric, succinic, oxalic, malic, glutamic, propionic,glycolic, gluconic, maleic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, β-hydroxybutyric, malonic, galactaric, galacturonic acid andthe like. Suitable pharmaceutically acceptable base addition saltsinclude, without limitation, metallic salts made from aluminum, calcium,lithium, magnesium, potassium, sodium and zinc or organic salts madefrom N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, lysine, procaine andthe like. Additional examples of pharmaceutically acceptable salts arelisted in Journal of Pharmaceutical Sciences (1977) 66:2. All of thesesalts may be prepared by conventional means from a modulator of FANCD2-containing foci by treating the compound with the appropriate acid orbase.

II. FANC D2 Foci

The cellular response to DNA damage is a complex interacting network ofpathways that mediate cell cycle checkpoints, DNA repair, and apoptosis.A model lesion for the investigation of these pathways has been DNAdouble-strand breaks, which rapidly induce cell cycle checkpoints andare repaired by a number of different pathways. In mammalian cells, bothhomologous recombination and nonhomologous recombination pathways areutilized. Extensive studies in mammalian cells have shown that complexesof DNA repair and cell cycle checkpoint proteins rapidly localize tosites of double-strand breaks induced by ionizing radiation. Theseproteins create foci that can be detected by immunofluorescent analyses.

The Fanconi anemia complementation group D2 (FANC D2) is a component ofa protein complex involved in chromosome stability and repair. Fanconianemia (FA) is a hereditary disorder characterized, in part, by adeficient DNA-repair mechanism that increases a person's risk for avariety of cancers. In response to DNA damage, the FA complex activatesFANC D2, which then associates with Breast Cancer, Type 1 polypeptide(BRCA1). Activation of FANC D2 occurs by phosphorylation of a serine 222residue by the Ataxia-Telangiectasia Mutated (ATM) kinase. In addition,activation via the FA pathway occurs via monoubiquitination of FANC D2at lysine 561. In its unmodified form, FANC D2 is diffusely locatedthroughout the nucleus. When ubiquitinated, FANC D2 forms dots, or foci,in the nucleus. The ubiquitination of FANC D2 and subsequent formationof nuclear foci occurs in response to DNA damage. Bycoimmunoprecipitation, Nakanishi et al. found constitutive interactionbetween FANC D2 and Nijmegen Breakage Syndrome 1 (NBS1), providingevidence that these proteins interact in two distinct assemblies tomediate S-phase checkpoint and resistance to mitomycin C-inducedchromosome damage (Nakashini et al., (2002) Nat Cell Biol. 4:913-20).

At least two types of ionizing radiation-induced foci have beenobserved: one containing the Rad5 1, BRCA1 and BRCA2 proteins, andanother containing the Mre11-Rad50-NBS1 complex. Rad51 foci, whichcontain the tumor suppressor proteins BRCA1 and BRCA2, also appearduring S phase in the absence of exogenous induction of DNA damage.

Mre11-Rad50-NBS1 foci can be detected as early as 10 min afterirradiation and are clearly present at sites of DNA breaks, while DNArepair is ongoing. These foci also colocalize with the BRCA1 protein,which has been shown to be required for their formation, possiblythrough its physical interaction with human Rad50 (hRad50). In addition,coimmunoprecipitation experiments performed with BRCA1 have indicatedthe presence of a large number of additional proteins in this complex(referred to as the BRCA1-associated surveillance complex). Theseinclude the mismatch repair proteins Msh2, Msh6, and Mlh1, thecheckpoint kinase ATM, the product of the Bloom's syndrome gene BLM, andreplication factor C. BRCA1, NBS1, and hMre11 have all been shown to besubstrates of the ATM kinase and to become phosphorylated in response tothe presence of DNA breaks.

The present invention is related to the discovery that cells exposed togenotoxic anti-neoplastic agents form FANC D2-containing foci. MultipleDNA damage response proteins have now been identified which form nuclearfoci, also called IRIFs (Ionizing-Radiation Inducible foci) in responseto DNA damage. Methods of detecting FANC D2-containing foci, as well asdetecting and quantitating the relative amount of ubiquitinated FANC D2polypeptide are described in U.S. application Ser. No. 10/165,099 andU.S. App. No. 60/540,380, the contents of which are incorporated intheir entirety herein by reference.

III. Means of Detecting FANC D2 Activation

1. Detection Using FANC D2-binding 7 Ligands

The total cellular level of FANC D2 protein does not significantlychange in response to DNA damage. Rather, DNA damage results inmonoubiquitination of FANC D2, as well as recruitment into FANCD2-containing foci. It will be appreciated by one skilled in the artthat an alternative to measuring the presence of FANC D2-containing fociis to use a ligand which specifically binds the monoubiquitinated, butnot the unubiquitinated form of FANC D2. To detect the presence ofmonoubiquitinated FANC D2, the ligand is preferably associated with adetectable label as described above. The main advantage of using such aligand, as will be appreciated by one skilled in the art, is that, dueto the typically low basal level of monoubiquitinated FANC D2 in cellswith undamaged DNA, the level of FANC D2-containing foci can be measuredin a sample taken from a living subject using the level ofmonoubiquitinated FANC D2 as a surrogate marker. An antibody whichspecifically recognizes the monoubiquitinated form of FANC D2 (FANCD2-L) has considerable utility as a rapid diagnostic. For instance, thisantibody could be used for:

-   -   1) Immunohistochemistry (IH). This antibody could be used to        examine tissue sections prepared from solid tumors (e.g.,        breast, ovarian, lung tumors). A positive signal by IH would        predict that the tumor will be resistant to cisplatin and        related drugs.    -   2) FACS analysis. Peripheral blood lymphocytes (PBLs) could be        screened with this antibody. A positive signal suggests the        presence of activated FANC D2, consistent with a recent exposure        of an individual to IR. or toxin. Thus, this antibody is a        useful extension of the radiation dosimeter assay described in        this application.    -   3) A high-throughput assay to screen for inhibitors of the        purified FA complex. These inhibitors will block the ability of        the FA complex to monoubiquitinate FANC D2 in vitro. The new        monoclonal antibody will be a useful reagent for end product        detection. Additional methods of measuring FANC D2-containing        foci using a ligand which specifically recognizes        monoubiquitinated FANC D2 include immunoblot analysis, or Enzyme        linked immunosorbant assays (ELISA) using extracts of samples        collected from living subjects, or FACS analysis (Harlow et al,        1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor        Laboratory Press, NY).

A sensitive measure of IR exposure is the increased monoubiquitinationof FANC D2. In undamaged cells, the ratio of FANC D2-L(monoubiquitinated isoform) to FANC D2-S (unubiquitinated isoform) isapproximately 0.5-0.6. This ratio (L/S) is readily calculated bycomparing the density of the L band to the S band on a western blot. Asensitive indicator of increased FANC D2 monoubiquitination and IRexposure is the conversion of the L/S ratio to 1.0 or greater.

2. Detection Using GFP-FANC D2 Fusion Proteins

An alternative approach for the detection of FANC D2 activation and fociformation is the use of a FANC D2 protein fused with a fluorescentprotein, for example, GFP. A functional fusion protein of FANC D2 andGFP is able to form foci upon exposure to genotoxic anti-neoplasticagents. These foci are then visible by fluorescence microscopy.Therefore, formation of FANC D2-containing foci can be measured as asurrogate marker for activation of the FA pathway in response toexposure to genotoxic anti-neoplastic agents. Methods of generating suchfusion protein constructs, as well as methods for detecting formation ofFANC D2-containing foci are described in U.S. App. No. 60/540,380, whichis incorporated herein by reference.

IV. Identifying Inhibitors of the FA Pathway

The present invention encompasses methods and compositions useful forthe treatment of neoplastic diseases using inhibitors of the FA pathway.Inhibitors of the FA pathway can be identified by methods describedherein, and also methods previously described, for example, in U.S.application Ser. No. 10/165,099 and U.S. App. No. 60/540,380, thecontents of which are incorporated herein by reference. For example,inhibitors of the FA pathway can be identified systematically using athree-tiered approach, as summarized in FIG. 1.

The first tier of screening comprises a high-throughput method toidentify agents which alter the formation of FANC D2-containing foci.Detection of FANC D2-containing foci, for example by using a FANC D2ligand such as anti-FANC D2 antibodies or cell lines expressing afunctional eGFP-FANC D2 fusion protein, are described in U.S.application Ser. No. 10/165,099 and U.S. App. No.60/540,380, thecontents of which are incorporated herein by reference. The methodcomprises contacting cells or a biological sample with a test compoundsimultaneously with, before or after exposure to a genotoxicanti-neoplastic agent, for example ionizing radiation (IR), mitomycin Cor cisplatin, at a dosage which induces formation of FANC D2-containingfoci. The number and size FANC D2-containing foci are then detected incells and compared with control cells which were not contacted with thetest compound. A decrease in the number and/or size of FANCD2-containing foci relative to control cells is indicative of an agentwhich is an inhibitor of the FA pathway, whereas an increase in thenumber and/or size of FANC D2-containing foci relative to control cellsis indicative of an agent which is an agonist of the FA pathway.Potential agonists and inhibitors thus identified can be further testedto determine whether they exert their effects directly on the FApathway, or act indirectly, for example, by directly causing damage toDNA (in the case of potential agonists of the FA pathway), or byreducing the effect of the genotoxic anti-neoplastic agent that was usedin the screen.

The second tier of screening involves the detection of ubiquitinatedFANC D2 polypeptides. As previously described, activation of the FApathway results in monoubiquitination of the FANC D2 polypeptide.Activation of the FA pathway can therefore be measured by detecting therelative amount of ubiquitinated FANC D2 compared with unubiquitinatedFANC D2 polypeptide. The ubiquitination of FANC D2 can be detected byperforming immunoblot analysis of protein extracts. Ubiquitinated FANCD2 migrates at a higher molecular weight band on immunoblot analyses,and can be detected using a labeled FANC D2 ligand, for example ananti-FANC D2 antibody. Therefore, the second tier of the screeningcomprises The method comprises contacting cells or a biological samplewith a test compound simultaneously with, before or after exposure to agenotoxic anti-neoplastic agent, for example ionizing radiation (IR),mitomycin C or cisplatin, at a dosage which induces formation of FANCD2-containing foci. The amount of ubiquitinated FANC D2 polypeptiderelative to unubiquitinated FANC D2 polypeptide is detected, andcompared with samples from control cells or biological samples whichwere not contacted with the test compound. A difference in the relativeamount of ubiquitinated FANC D2 relative to control cells indicates thatthe test compound is a modulator of the FA pathway. An increase in therelative amount of ubiquitinated FANC D2 polypeptide compared withcontrol cells or biological samples is indicative of an agonist of theFA pathway, whereas a decrease in the relative amount of ubiquitinatedFANC D2 polypeptide compared with control cells or biological samples isindicative of an inhibitor of the FA pathway. As described previously,the potential agonists and inhibitors thus identified can be furthertested to determine whether they exert their effects directly on the FApathway, or act indirectly, for example, by directly causing damage toDNA (in the case of potential agonists of the FA pathway), or byreducing the effect of the genotoxic anti-neoplastic agent that was usedin the screen.

The third tier of screening comprises in vitro testing of compounds forsensitivity to genotoxic anti-neoplastic agents. Contacting cells orbiological samples with inhibitors of the FA pathway would be expectedto increase the sensitivity of the samples/cells to genotoxicanti-neoplastic agents. Specific inhibition of the FA pathway by a testagent is expected to increase the sensitivity to a degree comparable to,for example, a cell line with a specific defect in one or morecomponents of the FA pathway. Cell lines useful for this type of assayinclude the ovarian cancer cell line, 2008, which is deficient in FANCF.2008 cells deficient in FANCF show heightened sensitivity to genotoxicanti-neoplastic agents (see graph in FIG. 8, open rectangle), and thissensitivity is restored to wild-type levels by overexpression of theFANCF (FIG. 8, open circle). The role of FANCF in restoring wild-typelevels of genotoxin sensitivity is then abolished by contacting with atest agent which inhibits the FA pathway (FIG. 8, closed circle), whileleaving the sensitivity to the genotoxic anti-neoplastic agentunaffected in the absence of the FANCF transfection (FIG. 8, closedrectangle).

The three tiers of screening described above provide a stream-linedapproach to rapidly identifying and characterizing potential modulatorsof the FA pathway. It should be understood that methods to identifymodulators are not limited to the particular embodiments of theinvention described above, and variations of the embodiments can be madeand still fall within the scope of the invention. In addition, the termsused herein are for the purpose of describing the particular embodimentsand are not intended to be limiting.

V. Inhibitors of the FA Pathway

The present invention contemplates the use of inhibitors of the FApathway. An inhibitor of the FA pathway includes any compound whichresults in the inhibition of formation of FANC D2-containing foci, whenadministered before, after or concomitantly with a genotoxicanti-neoplastic agent(s) which normally cause formation of FANCD2-containing foci. Examples of genotoxic anti-neoplastic agents whichinduce formation of FANC D2-containing foci include, but are not limitedto, ionizing radiation (IR) and DNA alkylating agents such as cisplatinor mitomycin C. Inhibition of the FA pathway can also be detected bymeasuring the relative amounts of ubiquitinated and unubiquitinated FANCD2 polypeptide of samples subjected to an agent which normally inducesubiquitination. Detection of FANC D2-containing foci using, for example,microscopic detection means, as well as determination of the relativeubiquitination state of the FANC D2 polypeptide, is described in U.S.Ser. No. 10/165099, filed Jun. 6, 2002, and U.S. Ser. No. 60/540380,filed Jan. 30, 2004, the contents of which are incorporated herein byreference. Briefly, FANC D2-containing foci can be detected usingimmunofluorescence microscopy, using anti-FANC D2 antibodies.Alternatively, a fluorescent protein-tagged version of FANC D2 can betransfected into the cells of interest, and formation of FANCD2-containing foci measured microscopically be detecting fluorescent‘foci’, again, as described in U.S. Ser. No. 60/540,380. Compounds whichinhibit the FA pathway, such as wortmannin and Trichostatin A, havepreviously been disclosed, for example in U.S. Ser. No. 60/540380, filedJan. 30, 2004.

The present invention describes additional examples of inhibitors of theFA pathway, including curcumin, H-9 and alsterpaullone, which wereidentified using the screening methods described herein.

H-9

-   -   Molecular weight 251.31    -   CAS Number 84468-17-7

H-9 kinase inhibitor, also known asN-2-Aminoethyl-5-Isoquinolinesulfonamide (formula I) is a knowninhibitor of several kinases, including PKA, PKG, PKC,Calcium/Calmodulin dependent protein kinase, and myosin light chainkinase (Inagaki et al., (1985) J Biol. Chem. 260(5):2922-5; Ito et al.,(1988) Int. J Immunopharmacol. 10:211-216)

Alsterpaullone

-   -   Molecular weight 293.27696    -   Molecular formula C16H11N3O3

Alsterpaullone (formula II), is known to inhibit Cdk1/cycline B, Gsk-3B,and Cdk5 (Sausville et al. (2000) Ann N Y Acad. Sci. 910:207-221;Schultz et al. (1999) J Med. Chem. 42:2909-2919).

Curcumin

-   -   CAS No.: 458-37-7    -   Molecular Weight: 368.39

Curcumin (Turmeric yellow, also known as 1,7-bis(4′-hydroxy-3′-methoxyphenyl)-1,6-heptadiene-3,5-dione,diferuloylmethane), a low molecular weight polyphenol derived from thespice, turmeric, is associated with regression of some solid tumors inhumans (Cheng et al. (2001) Anticancer Res. 21:2895-2900). Curcumin issafe in human trials at doses as high as 8000 mg/day (Cheng et al,ibid.). Recent studies suggest that curcumin may have activity in thetreatment of other human diseases, such as cystic fibrosis (Egan et al.(2004) Science 304:600-602).

Geldanamycin

-   -   CAS number: 30562-34-6    -   Molecular Weight: 560.6

Geldanamycin (formula IV) is a benzoquinone ansamycin antibiotic whichbinds to Hsp90 (Heat Shock Protein 90) and alters its function. Thepresent invention encompasses compositions and methods comprisinggeldanamycin and its analogs. Analogs of geldanamycin include17-(Allylamino)-17-demethoxy-geldanamycin (Schnur et al., (1995) J Med.Chem. 38:3806-12; Dunn (2002) J Natl. Cancer Inst 94, 1194-5). Thepresent invention further contemplates compositions and methodscomprising other inhibitors of HSP90, in particular benzoquinoneansamycin inhibitors of HSP90, coumarin derivatives (described, forexample, in WO 00/53169).

Other compounds which can inhibit the FA pathway include those compoundslisted within Table 2. Therefore, the inhibitor of the FA pathway can beselected from the group consisting of Alsterpaullone, (±)13-HODE,nifedipine, penitrem A, Geladanamycin, Go6976, leukotriene B3,Trichostatin-A, AG-370, Mitomycin C, Amanitin (alpha-amanitin),HNMPA-(AM)3, Propidium iodide, DRB, Ochratoxin, Ca-074-Me, K252c,Wortmannin, Actinomycin D, AG213, BAPTA-AM, Curcumin, Puromycin,Bumetanide, Methyladenine [3-methyladenine], H9, TPEN, spermine NONOate,PD00600, 5323069, and 1M556S.

VI. Inhibitors of Other DNA Damage Repair Pathways

Cells are continuously subjected to different kinds of DNA damage. Thesedamages can arise from exposure to a variety of internal and externalchemicals and radiation, including reactive oxygen species such assuperoxide (O₂ ⁻), hydrogen peroxide (H₂O₂). In addition, humans areconstantly exposed a vast array of carcinogens, many of which act bycausing damage to the DNA. It has been shown that at least six distinctmechanisms exist for DNA damage repair in humans, depending upon thetype of damage incurred.

Many cancers have a defect in at least one of the six major DNA damagerepair pathways. In addition to causing increased genomic instability,disruption of any of these DNA repair mechanisms can lead to increasedsensitivity to genotoxic anti-neoplastic agents. Therefore, thesecancers have increased dependence on one of the other five DNA damagerepair pathways for survival. Hence, disruption of a second, non-FA DNAdamage repair pathway in these neoplastic disorders, for example by asmall molecule inhibitor may result in selective cancer cell death.Stated differently, many cancers may turn out to have a dominant(primary) DNA damage repair pathway. Since one DNA damage repair pathwayis already abolished or significantly reduced in the cancer, an extraburden is placed on the dominant pathway in order to maintain the highproliferation rate and to prevent DNA damage of these cells. Disruptionof the dominant pathway in a cancer cell in which a major DNA damagerepair pathway is abolished or diminished, by means of an exogenousinhibitor, may therefore have a profound cytotoxic effect on the tumorcells but a relatively small cytotoxic effect on the surrounding normalcells.

Loss of the FA/BRCA pathway leads to chromosome instability, increasedcisplatin sensitivity, thus resulting in increased activity of theremaining non-FA DNA damage repair pathways, including the Base ExcisionRepair (BER) pathway. Accordingly, an inhibitor of a non-FA DNA damagerepair pathway, for example, BER (such as a PARP1 inhibitor or aninhibitor of a specific kinase in the BER pathway) would be lethal tothose cells, but may have little effect on normal (non-tumor) cells.

The present invention also contemplates the use of inhibitors of variousother DNA damage repair pathways. As previously described, there areseveral major pathways for DNA damage repair, including but not limitedto, non-homologous end joining (NHEJ), base excision repair (BER),nucleotide excision repair (NER), and mismatch repair (MR). Thesemechanisms are described, for example, in Hoeijmakers J H J (2001)Nature 411: 366-374, Svejstrup J Q (2002) Nat Rev Mol Cell Biol. 3:21-29, and in Panasci, DNA Repair in Cancer Therapy Humana Press, 2004,Totowa, N.J., which are incorporated herein by reference.

A. Non-Homologous End Joining (NHEJ)

DNA double strand breaks (DSBs) can be caused by any number ofenvironmental or other factors, including reactive oxygen species,ionizing radiation (IR) and certain anti-neoplastic drugs likebleomycin. Failure to repair DSBs can lead to a number of consequences,including mutations, chromosomal aberrations, and eventually cell death.Non-homologous end-joining (NHEJ), also called illegitimaterecombination, is one major pathway of repairing DSBs. Some members ofthe NHEJ pathway are shown in Table 1.

TABLE 1 Genes and Proteins Important for NEHJ Gene name Protein name;function MRE11 Exonuclease (3′ to 5′) NBS1 Mre11-interaction RAD50 Rolein stimulation of MRE11 exonuclease activity XRCC4 Unknown function;interacts with DNA ligase IV XRCC5 Ku80; forms heterodimer with Ku70which binds to DS DNA ends and DS/SS DNA junctions XRCC6 Ku70; formsheterodimer with Ku 70; deficiency correlated with elevated frequency ofT-cell lymphoma XRCC7 DNA-protein kinase; regulates Ku heterodimer

The DNA-dependent protein kinase (DNA-PK) consists of the catalyticsubunit (DNA-PKcs) and the regulatory subunit (the Ku70/Ku80heterodimer). The DNA-PKcs subunit is a serine/threonine kinase whichbelongs to the phosphatidyl inositol-3 kinase family. The Ku80/Ku70heterodimer (Ku) exhibits sequence-independent affinity fordouble-stranded termini and, upon binding to DNA, recruits and activatesthe DNA-PKcs catalytic subunit. Several candidate inhibitors of theDNA-PK have been described, for example viridins (Hanson, J. R. Nat.Prod. Rep., 12: 381-384, 1995), wortmannin, quercitins (Izzard et al.(1999) Cancer. Res., 59: 2581-2586), LY294002 (Vlahos et al. (1994) J.Biol. Chem., 269: 5241-5248), which are incorporated herein byreference. Other inhibitors of NHEJ include inhibitors of ATM disclosedwithin U.S. Ser. No.2004/0002492, which are incorporated herein byreference.

B. Base Excision Repair (BER)

Single Strand DNA breaks (SSBs) are one of the most frequent lesionsoccurring in cellular DNA. SSBs can occur spontaneously or asintermediates of enzymatic repair of base damage during Base ExcisionRepair (BER) (Caldecott (2001) Bioessays 23(5): 447-55). In this repairpathway, which follows the removal of a damaged base by a DNAglycosylase, the resulting apurinic/apyrimidinic (AP) site is processedfirst by the Ape1 AP endonuclease, leaving a 5′ deoxyribose-phosphate;then by an AP lyase activity leaving a 3′β-elimination product. Thesubsequent removal of these AP sites by DNA Polymerase β, or by aPCNA-dependent polymerase, allows the repair synthesis to fill-in eithera single nucleotide (for Pol β) or a longer repair patch (for Pol δ/ε),which are then re-ligated (Wilson (1998) Mutat Res. 407:203-15). If SSBsites are not efficiently processed and removed, clusters of damagedsites and stalled replication forks will form, resulting in theformation of DSBs with potentially lethal consequences for the cell(Chaudhry & Weinfeld (1997) J Biol Chem. 272:15650-5; Harrison, Hatahetet al. (1998) Nucleic Acids Res. 26:932-41).

Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger proteinthat catalyzes the transfer of ADP-ribose residues from NAD+ to itselfand different chromatin constituents, forming branched ADP-ribosepolymers. The enzymatic activity of PARP is induced upon DNA damage,suggesting a role of PARP in DNA repair and DNA damage-induced celldeath. Numerous inhibitors of PARP have been disclosed, some of whichare commercially available. For example, PJ-34N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide.Hcl, INHBP5-iodo-6-amino-1,2-benzopyrone, 3-Aminobenzamide, Benzamide,4-Amino-1,8-naphthalimide, 6(5H)-Phenanthridinone, 5-Aminoisoquinolinone(5-AIQ). hydrochloride, 4-Hydroxyquinazoline, 4-Quinazolinol,1,5-Isoquinolinediol, 5-Hydroxy-1(2H)-isoquinolinone,3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1 (2H)-isoquinolinone (DPQ) areall available from Inotek Pharmaceuticals (Beverly, Mass.). Othercompounds, such as GPI 15427 (Tentori et al. (2003) Proceedings of theAACR, 44, Abs No. 5466) and methoxyamine (Liuzzi et al., (1985) J. Biol.Chem. 260, 5252-5258; Rosa et al. (1991) Nucleic Acids Res., 19,5569-5574; and Horton et al. (2000) J. Biol. Chem., 275, 2211-2218) havebeen reported to enhance the anti-neoplastic efficacy of bothchemotherapy and radiation therapy.

C. Nucleotide Excision Repair (NER)

Nucleotide excision repair (NER) acts on a variety of helix-distortingDNA lesions, caused mostly by exogenous sources that interfere withnormal base pairing. The primary function of NER in man appears to bethe removal of damage, for example pyrimidine dimers, which are inducedby ultraviolet light (UV). Members of the NER pathway, defects of whichcan cause an autosomal recessive disease called xeroderma pigmentosum(XP), have been identified, including seven different genes, XPA, XPB,XPC, XPD, XPE, XPF and XPG, all of which function in the NER pathway(Hoeijmakers (2001) Mutat Res. 485:43-59).

Eukaryotic NER includes two major branches, transcription-coupled repair(TCR) and global genome repair (GGR) (de Laat et al. (1999) Genes Dev.13:768-85, Tornaletti & Hanawalt (1999) Biochimie. 81:139-46). GGR is aslow random process of inspecting the entire genome for injuries, whileTCR is highly specific and efficient and concentrates on damage-blockingRNA polymerase II. The two mechanisms differ in substrate specificityand recognition. In GGR, the XPC-HR23B complex recognizes damage locatedin nontranscribed regions (Sugasawa et al. (2001) Genes Dev. 15:507-21),whereas the arrest of RNA polymerase II (RNAPII) serves as therecognition signal in TCR. The molecular mechanism of RNAPIIdisplacement is currently unclear, but essential factors, such as theCocayne's syndrome proteins CSA, CSB, XPA-binding protein 2 (XAB2),TFIIH and XPG (Svejstrup 2002), have been identified to function in TCR.Subsequently, both in GGR and TCR, an open unwound structure formsaround the lesion. This creates specific cutting sites for XPG andERCC1-XPF nucleases, and the resulting gap is filled in byPCNA-dependent polymerase and sealed by DNA ligase (de Laat et al., id).

D. Mismatch Repair (MR)

Mismatch repair (MMR) removes both nucleotides mispaired by DNApolymerases and insertion/deletion loops caused by slippage duringreplication of repetitive sequences (Harfe & Jinks-Robertson (2000) AnnuRev Genet 34: 359-399). Initially, the heterodimeric MSH complexrecognizes the nucleotide mismatch, subsequently followed by interactionwith MLH1/PMS2 and MLH1/MLH3 complexes. Several proteins participate inprocess of the nucleotide excision and resynthesis. Tumor cellsdeficient in mismatch repair have much higher mutation frequencies thannormal cells (Parsons et al. (1993) Cell 75: 1227-1236, Bhattacharyya etal. (1994) Proc Natl Acad. Sci USA 91: 6319-6323). At least six genesMSH2, MLH1, PMS2, MSH3, MSH6 and MLH3 have been identified in humanswhich are involved in mismatch repair. Defects in these genes except forMSH3 leads to hereditary nonpolyposis colon cancer (HNPCC) (Hoeijmakers2001).

Other inhibitors to DNA damage repair have been disclosed, includingaphidicolin, (Gera (1993) J Immunol. 151:3746-57), rapamycin (mTORinhibitor, Sabers et al., (1995) J Biol. Chem. 270:815-22), the AGTinhibitor 06-benzylguanine (Bronstein et al., (1992) Cancer Res.52:3851-6).

VII. Identifying Inhibitors of Non-FA DNA Damage Repair Pathways.

As previously described, in certain situations the DNA damage repairpathways of the cell can be partially redundant. This presentsdifficulties in identifying agents which specifically block one pathway.Inhibitors identified using cell-based methods wherein the cells havefunctional DNA damage repair pathways may therefore have multipletargets, including in a plurality of DNA damage repair pathways.Therefore, use of cell lines deficient in one or more DNA damage repairpathways may greatly accelerate the identification of novel, specificinhibitors. Therefore, according to one aspect, a method of identifyingagents which inhibit a non-FA DNA damage repair pathway is provided. Themethod employs cells which have a lesion in the FA pathway. The methodcomprises contacting cells with an agent, and testing for sensitivity toa genotoxic anti-neoplastic agent. An agent which confers enhancedsensitivity to the genotoxic anti-neoplastic agent in test cellscontaining a lesion in the FA pathway when compared with control cellscontaining functional DNA damage repair pathways indicates that theagent inhibits a non-FA DNA damage repair pathway other than the pathwayin which the test cell contains a lesion. In one embodiment, test andcontrol cells are isogenic, except that the test cell contains a lesionin at least one component of the FA/BRCA pathway, for example, in FANCA,FANCB, FANCC, FANCD FANCE FANCF FANCG FANCL, and the ATR protein kinase,among others.

According to one embodiment, the method comprises comparing thesensitivities to genotoxic anti-neoplastic agents of two isogenic celllines which differ in the functionality of the FA pathway. In oneembodiment, two isogenic ovarian tumor lines, a parental 2008 and the2008 cells complemented with the FANCF cDNA, are employed. The parental2008 cells fail to express FANCF, these cells have a disruption of theFA pathway, and they are hypersensitive to cisplatin. Complementation ofthe 2008 cells with the FAN CF cDNA restores FA pathway in these cells.Therefore, these control cells therefore serve as a basis forcomparison. This isogenic pair of cells is subjected to a highthroughput chemical screen with a library of compounds, for examplekinase inhibitors. Agents which selectively kill 2008 cells (lacking theFA/BRCA pathway) but which do not kill the corrected 2008 plus FANCFcontrol cells are candidate inhibitors of a non-FA DNA damage repairpathway.

The availability of these isogenic cell lines also permits theidentification of gene products which are involved in DNA damage repairpathways other than the FA pathway. In one embodiment, genes affectingthe viability of the parental but not the control cells are tested bysystematic, mass inhibition using an siRNA library. For example, abar-coded siRNA library can be used to for stable transfection of thetwo cell lines. Genes that are required for viability of the 2008 cells,but not for the corrected cells. Genes which are important for DNAdamage repair pathways other than the FA pathway, for example in the BERpathway, is expected to have the result that siRNA knockdown of such agene will be lethal in the parental 2008 cells, but not in the control2008 cells which have been transfected with the FANCF cDNA.

Agents thus identified which can kill a cell in which one or more DNAdamage repair pathways is disrupted but do not kill an isogenic cellline in which the disruption is restored can be used in the treatment ofcancer. Disruption of two or more of the six major DNA damage repairpathways can result in cell death. Since many cancers already have theone pathway knocked out or repressed, a relatively non-toxic inhibitorof the second pathway, for example the BER pathway, may be sufficient tocause cytoreduction of the cancer, even in the absence of achemotherapeutic agent. In addition, in tumors cells in which the majorDNA damage repair pathways are intact, using two inhibitors incombination (e.g., one inhibitor of the FA pathway and one inhibitor ofthe BER pathway) may be sufficient to cause significant cytoreduction,provided that the toxicity of such a combination is not toxic to normal(non-cancer) cells. In such a case, a pro-drug strategy to enhanceuptake of these agents by cancer cells provide the necessary therapeuticindex.

VIII. Anti-Neoplastic Agents

Disclosed herein are methods of treating patients with neoplasticdisorders using a combination of anti-neoplastic agents in combinationwith inhibitors of DNA damage repair pathways. Anti-neoplastic agentswhich are particularly useful include, but are not limited to, agentswhich cause damage to the DNA. These agents include DNA alkylatingagents, intercalating agents, and the like. Further contemplated,therefore, is the use of DNA-damaging chemotherapeutic compoundsincluding, but not limited to, 1,3-Bis(2-Chloroethyl)-1-NitrosoUrea(BCNU), Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cyclophosphamide, Dacarbazine, Daunorubicin, Doxorubicin, Epirubicin,Etoposide, Idarubicin, Ifosfamide, Irinotecan, Lomustine,Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin,Temozolomide, and Topotecan. Furthermore, methods described herein canalso employ radiotherapeutic methods of treating neoplastic disorders.In one embodiment, the genotoxic anti-neoplastic agents do not inhibitDNA damage repair at the concentrations administered.

IX. Identifying Responders to Anti-neoplastic Agents

The present invention is based on the surprising discovery that theefficacy of the FA pathway of a cell strongly correlates with itssensitivity to chemotherapeutic agents. Therefore, in one aspect, theinvention provides a method of predicting whether a subject with aneoplastic disorder or disease will respond to a genotoxicanti-neoplastic agent. The method comprises obtaining a biologicalsample from the subject, and determining degree of ubiquitination of theFanconi anemia complementation group D2 (FANC D2) polypeptide within thebiological sample. A degree of ubiquitination of the FANC D2 polypeptidein the biological sample of the subject that is less than about 70% whencompared with a biological sample from a control subject is indicativeof a subject that will respond to a genotoxic anti-neoplastic agent.

In another aspect, the invention provides a method of predicting whethera subject with a neoplastic disorder or disease will respond to agenotoxic anti-neoplastic agent. The method comprises obtaining abiological sample from the subject, and determining the FANCD2-containing foci within the biological sample. A difference in fociformation, wherein the sample from the subject that contains less thanabout 70% of the FANC D2-containing foci when compared with thebiological sample from a control subject is indicative of a subject thatwill respond to a genotoxic anti-neoplastic agent.

In one embodiment, the neoplastic disorder is selected from the groupconsisting of leukemia, acute myeloid leukemia, chronic myeloidleukemia, chronic lymphatic leukemia, myelodysplasia, multiple myeloma,Hodgkin's disease or non-Hodgkin's lymphoma, small or non-small celllung carcinoma, gastric, intestinal or colorectal cancer, prostate,ovarian or breast cancer, head, brain or neck cancer, cancer in theurinary tract, kidney or bladder cancer, malignant melanoma, livercancer, uterine or pancreatic cancer.

According to these aspects, the ability of a biological sample toactivate the FA pathway, as determined by measuring the level of FANC D2monoubiquitination, is determined to identify responders tochemotherapeutic agents, particularly genotoxic anti-neoplastic agents.The anti-neoplastic agents can be any which are used for the treatmentof cancer, and in one embodiment, anti-neoplastic agents' mechanism ofaction is through the damage of DNA. These compounds include but are notlimited to: 1,3-Bis(2-Chloroethyl)-1-NitrosoUrea (BCNU), Busulfan,Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide,Dacarbazine, Daunorubicin, Doxorubicin, Epirubicin, Etoposide,Idarubicin, Ifosfamide, Irinotecan, Lomustine, Mechlorethamine,Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin, Temozolomide, andTopotecan and ionizing radiation.

In certain embodiments the patient or, alternatively, the biologicalsample obtained from the patient, can be exposed to the anti-neoplasticagent prior to determining the degree of ubiquitination of the FANC D2polypeptide. In one embodiment, the patient or biological sampleobtained from the subject is exposed at a dose that is less than orequal to the therapeutically effective dose. In another embodiment, theexposure is at 50% or less of the therapeutically. effective dose of theanti-neoplastic agent.

The degree of ubiquitination of the FANC D2 polypeptide can be comparedwith that of a control subject. As used herein, a control subject can bea single subject that has previously been determined to be normal withrespect to response to anti-neoplastic agents, or a number of normalsubjects. Biological samples from either a single control subject or anumber of control subjects can be used. In this aspect, a subject isdeemed to be a responder to an anti-neoplastic agent if the percentageof FANC D2 ubiquitination is less than about 70% when compared with asample from a subject, for example, less than 70%, less than 65%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10% or less, when compared with a sample from a subject thathas received the same or equivalent dose of anti-neoplastic agent as thetest sample. Furthermore, in embodiments involving exposure to ananti-neoplastic agent prior to determining the degree of ubiquitinationof the FANC D2 polypeptide, control samples can be prepared prior topreparation of the test samples, or prepared simultaneously topreparation of the test samples.

In one embodiment, the subject, or alternatively the biological sampletaken from the subject, can be treated with a genotoxic anti-neoplasticagent prior to measurement of the efficacy of the FA pathway. The dosageof the anti-neoplastic agent would be that necessary to induce the FApathway in a normal subject. Typically, the dosage of theanti-neoplastic agent would be from between about 5% to 100% of thetypical therapeutically effective dose, more typically between 20% to100%, and most typically between about 35%-100%.

As described herein, there are a number of ways in which to measure thedegree of ubiquitination of the FANC D2 polypeptide in biologicalsamples. The degree of ubiquitination of the FANC D2 polypeptide can bemeasured using immunoblot analysis as previously described.Alternatively, one could detect the formation of FANC D2-containingfoci, for example using immunofluorescence microscopy of biologicalsamples, as a surrogate marker for FANC D2 ubiquitination.

Subjects are considered responders if the formation of ubiquitinatedFANC D2 polypeptide is about 70% or less when compared with normalsubjects, for example 70% or less, 65% or less, 60% or less, 50% orless, 40% or less, 30% or less than in normal subjects.

X. Treatment of Neoplastic Disorders

In certain embodiments, a subject or patient is administered with atherapeutically effective dose of a genotoxic anti-neoplastic agent,simultaneously, before or after administration with an inhibitor of anon-FA DNA damage repair pathway, for example the FA pathway.Therapeutically effective dosages of many anti-neoplastic agents arewell-established, and can be found, for example, in Cancer Chemotherapyand Biotherapy: A Reference Guide Edition Number: 2 Tenenbaum, ed.Saunders & CO (1994) which is incorporated herein by reference.

Also provided herein are methods for treating a neoplastic disorder in asubject in need thereof. In one aspect, the method comprisesadministering to the subject an effective amount of an inhibitor of theFA pathway and a genotoxic anti-neoplastic agent. The anti-neoplasticagent can be selected from the group consisting of1,3-Bis(2-Chloroethyl)-1-NitrosoUrea (BCNU), Busulfan, Carboplatin,Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Dacarbazine,Daunorubicin, Doxorubicin, Epirubicin, Etoposide, Idarubicin,Ifosfamide, Irinotecan, Lomustine, Mechlorethamine, Melphalan, MitomycinC, Mitoxantrone, Oxaliplatin, Temozolomide, and Topotecan and ionizingradiation.

In another aspect, a method of treating a neoplastic disorder in asubject in need thereof is provided. The method comprises administeringto the subject an effective amount of an inhibitor of the FA pathway andan inhibitor of a non-FA DNA damage repair pathway. The inhibitor of anon-FA DNA damage repair pathway can be selected which inhibits any ofthe repair pathways, and can be selected from the group consisting ofPARP inhibitors, DNA-PK inhibitors, mTOR inhibitors, ERCC1 inhibitorsERCC3 inhibitors, ERCC6 inhibitors, ATM inhibitors, XRCC4 inhibitors,Ku80 inhibitors, Ku70 inhibitors, XPA inhibitors, CHK1 inhibitors, CHK2inhibitors, or pharmaceutically acceptable salts, esters, derivatives,solvates or prodrugs thereof. The inhibitor of the FA pathway can beadministered before, simultaneously with, or after administration of theinhibitor of the non-FA DNA damage repair pathway. The inhibitors can beadministered parenterally, orally or directly into the tumor.

The inhibitor of the FA pathway, as well as inhibitor of a non-FA DNAdamage repair pathway, can act to increase the sensitivity of aneoplastic disorder to a genotoxic anti-neoplastic agent. Therefore, inanother aspect, a method of increasing the sensitivity of a neoplasticdisorder to a genotoxic anti-neoplastic agent is provided. The methodcomprises administering before, after or concurrently with atherapeutically effective dose of the agent a combination of aneffective amount of an inhibitor of the FA pathway and an inhibitor of anon-FA DNA damage repair pathway. The method can be useful for thetreatment of many types of neoplastic disorders, and can be selectedfrom the group consisting of leukemia, acute myeloid leukemia, chronicmyeloid leukemia, chronic lymphatic leukemia, myelodysplasia, multiplemyeloma, Hodgkin's disease or non-Hodgkin's lymphoma, small or non-smallcell lung carcinoma, gastric, intestinal or colorectal cancer, prostate,ovarian or breast cancer, head, brain or neck cancer, cancer in theurinary tract, kidney or bladder cancer, malignant melanoma, livercancer, uterine or pancreatic cancer.

The inhibitors of the FA pathway are further useful as agents whichincrease the sensitivity of a neoplastic disorder to a genotoxicanti-neoplastic agent. Therefore, in another aspect, the inventionprovides a method of increasing the sensitivity of a neoplastic disorderto a genotoxic anti-neoplastic agent. The method comprises administeringbefore, after or concurrently with a therapeutically effective dose ofan genotoxic anti-neoplastic agent, an effective amount of an inhibitorof the FA pathway. As previously described, the inhibitor of the FApathway can be administered before, simultaneously with, or afteradministration of the inhibitor of the non-FA DNA damage repair pathway,and can be administered parenterally, orally or directly into the tumor.In one embodiment, the method further comprises administering aninhibitor of a non-FA DNA damage repair pathway, in addition to the FAinhibitor and genotoxic anti-neoplastic agent. The inhibitor of thenon-FA DNA damage repair pathway can be administered before, after, orconcurrently with a therapeutically effective dose of the FA pathwayinhibitor and genotoxic anti-neoplastic agent.

The efficacy of compositions disclosed herein in preventing or treatingneoplastic disorders can be tested, for example, in animal models ofspecific neoplastic disorders. Numerous examples of animal models arewell known to those skilled in the art, and are disclosed, for example,in Holland, Mouse Models of Cancer (Wiley-Liss 2004); Teicher, TumorModels in Cancer Research (Humana Press; 2001); Kallman, Rodent TumorModels in Experimental Cancer Therapy (Mcgraw-Hill, TX, 1987); Hedrich,The Laboratory Mouse (Handbook of Experimental Animals) (Academic Press,2004); and Arnold and Kopf-Maier, Immunodeficient Animals: Models forCancer Research (Contributions to Oncology, Vol 51) (Karger, 1996), thecontents of which are incorporated herein in their entirety.

XI. Test Compounds According to the Invention

Whether in an in vitro or in vivo system, the invention encompassesmethods by which to screen compositions which can inhibit the formationof FANC D2-containing foci, as well as compositions which inhibit DNAdamage repair pathways other than the FA pathway. Candidate modulatorcompounds from large libraries of synthetic or natural compounds can bescreened. Numerous means are currently used for random and directedsynthesis of saccharide, peptide, and nucleic acid based compounds.Synthetic compound libraries are commercially available from a number ofcompanies including Maybridge Chemical Co. (Trevillet, Cornwall, UK),Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), andMicrosource (New Milford, Conn.). A rare chemical library is availablefrom Aldrich (Milwaukee, Wis.). Combinatorial libraries are availableand can be prepared. Alternatively, libraries of natural compounds inthe form of bacterial, fungal, plant and animal extracts are availablefrom e.g., Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or arereadily producible by methods well known in the art. Additionally,natural and synthetically produced libraries and compounds are readilymodified through conventional chemical, physical, and biochemical means.

Useful compounds may be found within numerous chemical classes, thoughtypically they are organic compounds, including small organic compounds.Small organic compounds have a molecular weight of more than 50 yet lessthan about 2,500 Daltons, preferably less than about 750, morepreferably less than about 350 Daltons. Exemplary classes includeheterocycles, peptides, saccharides, steroids, and the like. Thecompounds may be modified to enhance efficacy, stability, pharmaceuticalcompatibility, and the like. Structural identification of an agent maybe used to identify, generate, or screen additional agents. For example,where peptide agents are identified, they may be modified in a varietyof ways to enhance their stability, such as using an unnatural aminoacid, such as a D-amino acid, particularly D-alanine, by functionalizingthe amino or carboxylic terminus, e.g., for the amino group, acylationor alkylation, and for the carboxyl group, esterification oramidification, or the like.

Candidate modulators which may be screened according to the methods ofthe invention include receptors, enzymes, ligands, regulatory factors,and structural proteins. Candidate modulators also include nuclearproteins, cytoplasmic proteins, mitochondrial proteins, secretedproteins, plasmalemma-associated proteins, serum proteins, viralantigens, bacterial antigens, protozoan antigens and parasitic antigens.Candidate modulators additionally comprise proteins, lipoproteins,glycoproteins, phosphoproteins and nucleic acids (e.g., RNAs such asribozymes or antisense nucleic acids). Proteins or polypeptides whichcan be screened using the methods of the present invention includehormones, growth factors, neurotransmitters, enzymes, clotting factors,apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumorsuppressors, structural proteins, viral antigens, parasitic antigens,bacterial antigens and antibodies (see below).

Candidate modulators which may be screened according to the inventionalso include substances for which a test cell or organism might bedeficient or that might be clinically effective in higher-than-normalconcentration as well as those that are designed to eliminate thetranslation of unwanted proteins. Nucleic acids of use according to theinvention not only may encode the candidate modulators described above,but may eliminate or encode products which eliminate deleteriousproteins. Such nucleic acid sequences are antisense RNA and ribozymes,as well as DNA expression constructs that encode them. Note thatantisense RNA molecules, ribozymes or genes encoding them may beadministered to a test cell or organism by a method of nucleic aciddelivery that is known in the art, as described below. Inactivatingnucleic acid sequences may encode a ribozyme or antisense RNA specificfor the target mRNA. Ribozymes of the hammerhead class are the smallestknown, and lend themselves both to in vitro production and delivery tocells (summarized by Sullivan, (1994) J. Invest. Dermatol., 103:85S-98S; Usman et al., (1996), Curr. Opin. Struct. Biol., 6: 527-533).

XII. Pharmaceutical Compositions

In another aspect, the invention relates to methods and pharmaceuticalcompositions comprising an inhibitor of the FA pathway in combinationwith an anti-neoplastic agent and/or inhibitor of a non-FA DNA damagerepair pathway, as described in the preceding section, and apharmaceutically acceptable carrier, as described below. Thepharmaceutical composition comprising an inhibitor of the FA pathway isuseful for treating a variety of diseases and disorders includingcancer, and may be useful as protective agents against genotoxicanti-neoplastic agents.

In one embodiment, the invention provides for a method of treating aneoplastic disorder in a subject in need thereof comprisingadministering a combination of an effective amount of:

a) an inhibitor of the FA pathway or pharmaceutically acceptable salts,esters, derivatives, solvates or prodrugs thereof, and

b) a genotoxic anti-neoplastic agent.

Examples of inhibitors of the FA pathway include H-9, alsterpaullone andcurcumin. However, it will be appreciated by those skilled in the artthat additional inhibitors of the FA pathway can be identified, forexample, using the methods described herein. In this regard, aninhibitor of the FA pathway can be a small molecule, and antibody, aribozyme or siRNA molecule.

The method can be used in the treatment of various neoplastic disorders,including leukemia, acute myeloid leukemia, chronic myeloid leukemia,chronic lymphatic leukemia, myelodysplasia, multiple myeloma, Hodgkin'sdisease or non-Hodgkin's lymphoma, small or non-small cell lungcarcinoma, gastric, intestinal or colorectal cancer, prostate, ovarianor breast cancer, head, brain or neck cancer, cancer in the urinarytract, kidney or bladder cancer, malignant melanoma, liver cancer,uterine or pancreatic cancer. In one embodiment, the method is used totreat ovarian cancer.

The dosage of the inhibitor of the FA pathway depends on severalfactors, including solubility, bioavailability, plasma protein binding,kidney clearance, and inhibition constants. In certain therapeuticapplications, an adequate amount to accomplish at least partialinhibition of the FA pathway is defined as an “effective dose”. Amountsneeded to achieve this dosage will depend upon the severity of thedisease and the general state of the patient's own immune system, butgenerally range from 0.005 to 5.0 mg of the inhibitor per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. Alternatively, the dosage can be administered using a functionaldosage, since the activation of the FA pathway in a subject can bedetermined empirically using the ubiquitination of the FANC D2polypeptide using the methods described herein. Therefore, an “effectivedose” of an inhibitor of the FA pathway can mean a dose required toreduce the level of FANC D2 ubiquitination to about 70% or less whencompared with a control sample, more typically to about 50% or less thana control sample. In this regard, a control sample is ideally taken fromthe same subject, before administration of the inhibitor.

The dosage of the inhibitor of the FA pathway in relation to the dosageof the genotoxic anti-neoplastic agent can be expressed as a ratio. Theinhibitor of the FA pathway can be administered at a ratio of betweenabout 100:1 to about 1:100, on a molar basis, in relation to thegenotoxic anti-neoplastic agent, for example, at 1:100, 1:50, 1:10, 1:5,1:2, 1:1, 2:1, 5:1, 10:1, 20:1, 50:1, or 100:1.

The genotoxic anti-neoplastic agent are agents which are used to treatneoplastic disorders, and include 1,3-Bis(2-Chloroethyl)-1-NitrosoUrea(BCNU), Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cyclophosphamide, Dacarbazine, Daunorubicin, Doxorubicin, Epirubicin,Etoposide, Idarubicin, Ifosfamide, Irinotecan, Lomustine,Mechlorethamine, Melphalan, Mitomycin C, Mitoxantrone, Oxaliplatin,Temozolomide, and Topotecan.

Dosages of the anti-neoplastic agents listed above have been wellestablished for different types of neoplastic disorders. However,co-administration with inhibitors of the FA pathway can increase thesensitivity of the neoplastic disorders to the anti-neoplastic agents.Therefore, it is possible that the dosage of the anti-neoplastic agentswill be less than is typically administered for the given neoplasticdisorder. The lower dosage may have the additional advantage of reducedside effects. However, typically, the dosage of the anti-neoplasticagent is expected to be within about 20%-100% of the typical dosage forthe given neoplastic disorder, more typically between about 35%-100%.

In yet another embodiment, the present invention provides for a methodof treating a neoplastic disorder in a subject in need thereof,comprising administering to the subject a combination of an effectiveamount of:

(a) an inhibitor of the FA pathway or pharmaceutically acceptable salts,esters, derivatives, solvates or prodrugs thereof, and

(b) an inhibitor of a DNA damage repair pathway.

The inhibitor of a DNA damage repair pathway can be selected from thegroup consisting of PARP inhibitors, DNA-PK inhibitors, FA inhibitors,mTOR inhibitors, ERCC1 inhibitors, ERCC3 inhibitors, ERCC6 inhibitors,ATM inhibitors, XRCC4 inhibitors, Ku80 inhibitors, Ku70 inhibitors, XPAinhibitors, CHK1 inhibitors, CHK2 inhibitors, or pharmaceuticallyacceptable salts, esters, derivatives, solvates or prodrugs thereof.

In one embodiment, the non-FA DNA damage repair pathway is a pathwayother than the FA pathway. In one embodiment, the inhibitor targets apathway selected from the group consisting of the non-homologous endjoining DNA damage repair pathway, the mismatch repair pathway, and thenucleotide excision pathway. In another embodiment, the inhibitortargets the non-homologous end joining DNA damage repair pathway. In yetanother embodiment, the inhibitor targets the direct reversal pathway.In another embodiment, the inhibitor targets the mismatch repairpathway. In still another embodiment, the inhibitor targets thenucleotide excision repair pathway. In another embodiment, the inhibitortargets the base excision repair pathway.

Ideal dosages of the inhibitor of a DNA damage repair pathway, asdescribed above for inhibitors of the FA pathway, will depend upon theseverity of the disease and the general state of the patient's ownimmune system, but generally range from 0.005 to 5.0 mg of the inhibitorper kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose beingmore commonly used. Alternatively, the appropriate dosage can bedetermined empirically, inhibition of DNA damage repair pathways can bemeasured using biological samples taken from the subject. Therefore, an“effective dose” of an inhibitor of the DNA damage repair pathway canmean a dose required to reduce the level of the specific pathway toabout 70% or less when compared with a control sample, more typically toabout 50% or less than a control sample. In this regard, a controlsample is ideally taken from the same subject, before administration ofthe inhibitor.

In yet another embodiment, the present invention provides for a methodof treating a neoplastic disorder in a subject in need thereof,comprising administering to said subject a combination of an effectiveamount of:

(a) an inhibitor of the FA pathway or pharmaceutically acceptable salts,esters, derivatives, solvates or prodrugs thereof,

(b) an inhibitor of a non-FA DNA damage repair pathway, and

(c) a genotoxic anti-neoplastic agent or pharmaceutically acceptablesalts, esters, derivatives, solvates or prodrugs thereof.

The inhibitor of the FA pathway, its dosage and method ofadministration, are as described previously. Likewise, the inhibitor ofa non-FA DNA damage repair pathway, as well as its dosage and method ofadministration are the same as previously described. However, aspreviously described, administration of inhibitors of the FA pathway, aswell as of a non-FA DNA damage repair pathway, can heighten thesensitivity to a genotoxic anti-neoplastic agent. Therefore, it ispossible that the dosage of the anti-neoplastic agents will be less thanis typically administered for the given neoplastic disorder. The lowerdosage may have the additional advantage of reduced side effects.However, typically, the dosage of the anti-neoplastic agent is expectedto be within about 20%-100% of the typical dosage for the givenneoplastic disorder, more typically between about 35%-100%.

The compounds of the present invention, or pharmaceutically acceptablesalts, esters, derivatives, solvates or prodrugs thereof, can beformulated for oral, intravenous, intramuscular, subcutaneous, topicaland/or parenteral administration for the therapeutic or prophylactictreatment of diseases. For oral or parental administration, compounds ofthe present invention can be mixed with conventional pharmaceuticalcarriers and excipients and used in the form of tablets, capsules,elixirs, suspensions, syrups, wafers and the like. The compositionscomprising a compound of this present invention will contain from about0.1% to about 99.9%, about 1% to about 98%, about 5% to about 95%, about10% to about 80% or about 15% to about 60% by weight of the activecompound.

The compounds of the present invention can be administered at separatetimes, using separate methods of administration. For example, in certainsituations, it may be advantageous to administer the inhibitor of the FApathway before, simultaneously with, or after administration of thegenotoxic anti-neoplastic agent or other agents. Likewise, the method ofadministration of each compound will depend on the optimal means ofadministration thereof.

The pharmaceutical preparations disclosed herein are prepared inaccordance with standard procedures and are administered at dosages thatare selected to reduce, prevent, or eliminate cancer, or to provide aprotective effect against genotoxic anti-neoplastic agents such asionizing radiation. (See, e.g., Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa.; and Goodman and Gilman,Pharmaceutical Basis of Therapeutics, Pergamon Press, New York, N.Y.,the contents of which are incorporated herein by reference, for ageneral description of the methods for administering variousantimicrobial agents for human therapy). The compositions of the presentinvention can be delivered using controlled (e.g., capsules) orsustained release delivery systems (e.g., biodegradable matrices).Examples of delayed release delivery systems for drug delivery suitablefor administering compositions of the invention are described in U.S.Pat. No. 4,452,775, U.S. Pat. No. 5,239,660, and U.S. Pat. No.3,854,480.

The pharmaceutically acceptable compositions of the present inventioncomprise one or more compounds of the present invention in associationwith one or more non-toxic, pharmaceutically acceptable carriers and/ordiluents and/or adjuvants and/or excipients, collectively referred toherein as “carrier” materials, and if desired other active ingredients.The compositions may contain common carriers and excipients, such ascorn starch or gelatin, lactose, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid.The compositions may contain crosarmellose sodium, microcrystallinecellulose, sodium starch glycolate and alginic acid.

Tablet binders that can be included are acacia, methylcellulose, sodiumcarboxymethylcellulose, polyvinylpyrrolidone (Providone), hydroxypropylmethylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearate or other metallicstearates, stearic acid, silicon fluid, talc, waxes, oils and colloidalsilica.

Flavoring agents such as peppermint, oil of wintergreen, cherryflavoring or the like can also be used. It may also be desirable to adda coloring agent to make the dosage form more aesthetic in appearance orto help identify the product comprising a compound of the presentinvention.

For oral use, solid formulations such as tablets and capsules areparticularly useful. Sustained released or enterically coatedpreparations may also be devised. For pediatric and geriatricapplications, suspension, syrups and chewable tablets are especiallysuitable. For oral administration, the pharmaceutical compositions arein the form of, for example, a tablet, capsule, suspension or liquid.The pharmaceutical composition is preferably made in the form of adosage unit containing a therapeutically-effective amount of the activeingredient. Examples of such dosage units are tablets and capsules. Fortherapeutic purposes, the tablets and capsules which can contain, inaddition to the active ingredient, conventional carriers such as bindingagents, for example, acacia gum, gelatin, polyvinylpyrrolidone,sorbitol, or tragacanth; fillers, for example, calcium phosphate,glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants, forexample, magnesium stearate, polyethylene glycol, silica or talc:disintegrants, for example, potato starch, flavoring or coloring agents,or acceptable wetting agents. Oral liquid preparations generally are inthe form of aqueous or oily solutions, suspensions, emulsions, syrups orelixirs and may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous agents, preservatives, coloringagents and flavoring agents. Examples of additives for liquidpreparations include acacia, almond oil, ethyl alcohol, fractionatedcoconut oil, gelatin, glucose syrup, glycerin, hydrogenated edible fats,lecithin, methyl cellulose, methyl or propyl para-hydroxybenzoate,propylene glycol, sorbitol, or sorbic acid.

For intravenous (iv) use, compounds of the present invention can bedissolved or suspended in any of the commonly used intravenous fluidsand administered by infusion. Intravenous fluids include, withoutlimitation, physiological saline or Ringer's solution.

Formulations for parental administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions or suspensions can be prepared from sterile powders orgranules having one or more of the carriers mentioned for use in theformulations for oral administration. The compounds can be dissolved inpolyethylene glycol, propylene glycol, ethanol, corn oil, benzylalcohol, sodium chloride, and/or various buffers.

For intramuscular preparations, a sterile formulation of compounds ofthe present invention or suitable soluble salts forming the compound,can be dissolved and administered in a pharmaceutical diluent such asWater-for-Injection (WFI), physiological saline or 5% glucose. Asuitable insoluble form of the compound may be prepared and administeredas a suspension in an aqueous base or a pharmaceutically acceptable oilbase, e.g. an ester of a long chain fatty acid such as ethyl oleate.

For topical use the compounds of present invention can also be preparedin suitable forms to be applied to the skin, or mucus membranes of thenose and throat, and can take the form of creams, ointments, liquidsprays or inhalants, lozenges, or throat paints. Such topicalformulations further can include chemical compounds such asdimethylsulfoxide (DMSO) to facilitate surface penetration of the activeingredient.

For application to the eyes or ears, the compounds of the presentinvention can be presented in liquid or semi-liquid form formulated inhydrophobic or hydrophilic bases as ointments, creams, lotions, paintsor powders.

For rectal administration the compounds of the present invention can beadministered in the form of suppositories admixed with conventionalcarriers such as cocoa butter, wax or other glyceride.

Alternatively, the compound of the present invention can be in powderform for reconstitution in the appropriate pharmaceutically acceptablecarrier at the time of delivery. In another embodiment, the unit dosageform of the compound can be a solution of the compound or a salt thereofin a suitable diluent in sterile, hermetically sealed ampoules.

The amount of the compound of the present invention in a unit dosagecomprises a therapeutically-effective amount of at least one activecompound of the present invention which may vary depending on therecipient subject, route and frequency of administration. A subjectrefers to an animal such as an ovine or a mammal, including a human.

According to this aspect of the present invention, the novelcompositions disclosed herein are placed in a pharmaceuticallyacceptable carrier and are delivered to a recipient subject (including ahuman subject) in accordance with known methods of drug delivery. Ingeneral, the methods of the invention for delivering the compositions ofthe invention in vivo utilize art-recognized protocols for deliveringthe agent with the only substantial procedural modification being thesubstitution of the compounds of the present invention for the drugs inthe art-recognized protocols.

The compounds of the present invention provide a method for treatingpre-cancerous or cancerous conditions, or for use as a protective agentagainst genotoxic anti-neoplastic agents. As used herein, the term “unitdosage” refers to a quantity of a therapeutically effective amount of acompound of the present invention that elicits a desired therapeuticresponse. The term “treating” is defined as administering, to a subject,a therapeutically effective amount of at least one compound of thepresent invention, both to prevent the occurrence of a pre-cancer orcancer condition, or to control or eliminate pre-cancer or cancercondition. The term “desired therapeutic response” refers to treating arecipient subject with a compound of the present invention such that apre-cancer or cancer condition is reversed, arrested or prevented in arecipient subject.

The compounds of the present invention can be administered as a singledaily dose or in multiple doses per day. The treatment regime mayrequire administration over extended periods of time, e.g., for severaldays or for from two to four weeks. The amount per administered dose orthe total amount administered will depend on such factors as the natureand severity of the disease condition, the age and general health of therecipient subject, the tolerance of the recipient subject to thecompound and the type of cancer, the sensitivity of the cancer totherapeutic agents, and, if used in combination with other therapeuticagent(s), the dose and type of therapeutic agent(s) used.

A compound according to this invention may also be administered in thediet or feed of a patient or animal. The diet for animals can be normalfoodstuffs to which the compound can be added or it can be added to apremix.

The compounds of the present invention may be taken in combination,together or separately with any known clinically approved agent to treata recipient subject in need of such treatment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. Further disclosure relevant to themethods and embodiments described herein can be found in Chirnomas etal., Mol. Cancer Ther. 5(4):952-961 (2006) and Taniguchi and D'Andrea,published electronically in Blood (2006) at DOI10.1182/blood-2005-10-4240, both of which are incorporated by referencein their entirety. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

EXAMPLES Example 1 Methods

Cell Lines and Cell Culture.

HeLa cells, PD20 (FA-D2) fibroblasts, and GM6914 (FA-A) fibroblasts weregrown as previously described (Taniguchi et al. (2002) Cell.109:459-472). Briefly, cells were grown in Dulbecco's modified eaglesmedium (DMEM) supplemented with 15% fetal calf serum (FCS). TheFANCF-deficient ovarian tumor line (2008) and FANCF cDNA corrected 2008cells were previously described. Breast cancer cell line MCF7 waspurchased from the American Type Culture Collection (Manassas, Va.).OVCAR5 and OVCAR8 were grown as previously described.

Plasmids and Retroviral Infection

The retroviral expression vector, pMMP-puro (Ory et al., (1996) ProcNatl Acad. Sci USA. 93:11400-11406) and pMMP-puro-FANCD2 was describedpreviously (Timmers et al., (2001) Mol Cell. 7:241-248; Garcia-Higueraet al., (2001) Mol Cell. 7:249-262). pMMPpuro EGFP-FANCD2 wasconstructed by adding EGFP cDNA sequence (from pEGFP-N1 (Clontech)) tothe N-terminus of the FANCD2 cDNA sequence. The cDNA insert was verifiedby direct DNA sequencing. Production of pMMP retroviral supernatants andinfection of fibroblasts (PD20 fibroblasts) were performed as previouslydescribed (Naf et al., (1998) Mol Cell Biol, 18:5952-5960). After 48hours, cells were trypsinized and selected in medium containingpuromycin (1 μg/mL). Dead cells were removed, and surviving cells weregrown under continuous selection in puromycin. Subcloning of PD20fibroblasts infected with pMMPpuroEGFP-FANCD2 was performed by limiteddilution, and a clone which showed clear EGFP-FANCD2 foci formation inresponse to IR treatment (15Gy, 10 hr) was selected (PD20F-EGFP-FANCD2clone 7) for the drug screening study.

Cytotoxicity Assays.

Human cells (HeLa cells, PD20 fibroblasts, or 2008 cells, whereindicated; Taniguchi et al., (2003) Nat Med. 9:568-574) were seeded onto12-well plates at 9×10⁴ cells/well in DMEM-15% FCS (5 ml). After cellsattached for 16 to 24 h, the medium was replaced with DMEM-15% FCScontaining cisplatin (CDDP) or MMC (Sigma) at various concentrations,with or without a kinase inhibitor or curcumin (Sigma) at eithervariable or the same concentration. The cells were incubated at 37° C.for one day. The media was removed and the wells were washed once withphosphate-buffered saline (PBS) before the addition of fresh DMEM-15%FCS. After incubation for 5 to 6 days, monolayers were washed twice with(PBS) before and fixed for 5 to 10 min at 23° C. in 10% (vol/vol)methanol and 10% (vol/vol) acetic acid. Adherent colonies were stainedfor 2 to 10 min at 23° C. with 1% (wt/vol) crystal violet (Sigma) inmethanol (0.5 ml per well). Plates were rinsed in distilled water, andthe adsorbed dye was resolubilized with methanol containing 0.1%(wt/vol) sodium dodecyl sulfate SDS (0.5 ml per well) by gentleagitation for 1 to 4 h at 23° C. Dye solution (150 μl) was transferredto 96-well plates and diluted (1:3) in methanol. Crystal violetconcentrations were measured photometrically (595 nm) in a model 3550microplate reader (Bio-Rad). For quantitation, readings of opticaldensity at 595 nm were normalized to those obtained from untreated cells(concentration of CDDP=0 nM), assumed to yield 100% cell survival.

Immunofluorescence Microscopy.

Cells were seeded onto four-well chamber slides (Falcon) and cultivatedfor 16 to 24 h. Slides were rinsed with PBS, and adherent cells werefixed for 20 min at 23° C. in paraformaldehyde (4% [wt/vol] in PBS) andpermeabilized with Triton X-100 (0.3% [vol/vol] in PBS) for 10 min at23° C. Staining with primary (affinity-purified anti-FANCD2) andsecondary (fluorescein-conjugated goat anti-rabbit) antibodies was for 2h at 23° C., followed by counterstaining for 5 min at 23° C. with DAPI(4′,6-diamidino-2-phenylindole dihydrochloride; 10 μg/ml in PBS; Sigma).Slides were mounted in Vectashield (Vector Laboratories) and analyzed byfluorescence microscopy.

High Throughput Screen for Small Molecule Inhibitors of the FA/BRCAPathway.

Initially, we transduced PD20 (FA-D2) fibroblasts with thepMMP-GFP-FANCD2-puro retroviral supernatant and twenty individualpuromycin resistant colonies were selected. One clone (clone 7) had alow level of expression of GFP-FANCD2 in the nucleus, judged byfluorescence microscopy, but formed bright GFP foci in response to IR(2Gy). Clone 7 was used in subsequent experiments.

For high throughput screens, clone 7 cells were plated in 384 wellplates. One hour after plating, a chemical compound, from a commerciallibrary (Kau et al., (2003) Cancer Cell. 4:463-476.), was added to eachwell, at a single concentration of approximately 20-40 micromolar.Library compounds were added to duplicate plates. After a twelve-hourincubation, the plates were irradiated (15 Gy), and following anadditional twelve-hour incubation, cells were fixed for GFP microscopy.Photomicrographs were obtained for each well, and wells with significant(50%) reduction in GFP foci, were identified by visual inspection.

Immunoblotting.

Whole cell lysates were electrophoresed by SDS-PAGE (7% polyacrylamide,bisacrylamide gels for ATR and FANCD2 analysis and 10% for Chk1 and RPA1analysis). Proteins were transferred to nitrocellulose, blocked, andincubated with primary antibodies as described (Andreassen et al.,(2004) Genes Dev. 18:1958-1963). Antibodies included anti-FANCD2 (E35,1:1000; Garcia-Higuera et al., id), anti-ATR (1:1000 Santa Cruz),anti-phospho-S345-Chk1 (1:1000, Cell Signaling), and anti-Chk1 (1:500Santa Cruz). Membranes were washed, incubated with HRP-linked secondaryantibodies (Amersham), and detected by chemiluminescence (Amersham) asdescribed (Taniguchi et al., (2002) Cell. 109:459-472).

In vitro Kinase Assay for ATR.

The in vitro kinase assay with Flag-ATR and kinase-dead Flag-ATR hasbeen previously described (Andreassen et al., id).

Example 2 Identification and Characterization of Potential Inhibitors ofFANC D2 Ubiquitination and Foci Formation

Using the microscopy methods detailed above, the 489 known bioactivecompounds within the collection of the Institute of Chemistry and CellBiology (ICCB), Harvard Medical, were screened for inhibition ofIR-mediated FANC D2 foci formation. A number of positives wereidentified using the primary screen, which employed high throughputfluorescence microscopy to identify agents which blocked the formationof FANC D2-containing foci upon exposure to ionizing radiation. A numberof compounds were identified, as described within Table 2.

TABLE 2 Compounds identified through screen of ICCB Bioactives CompoundKnown function Alsterpaullone cdk1/cycB inhibitor (+−)13-HODE bioactivelipid NIFEDIPINE calcium channels PENITREM A potassium channelsGeladanamycin HSP90 inhibitor Go6976 PKC inhibitor LEUKOTRIENE B3bioactive lipid Trichostatin-A histone deacetylase inhibitor AG-370 PDGFreceptor kinase inhibitor Mitomycin C DNA crosslinker Amanitin(alpha-amanitin) transcription inhibitor HNMPA-(AM)3 Insulin receptor TKinhibitor Propidium iodide DNA intercalator DRB CK II inhibitorOchratoxin stimulates ER Ca²⁺ATPase Ca-074-Me Cathepsin B inhibitorK252c PKC inhibitor Wortmannin PI3 kinase, other kinases inhibitorActinomycin D transcription inhibitor AG213 EGF-R inhibitor BAPTA-AMcell permeable Ca²⁺chelator Curcumin Inductor of apoptosis in cancercells Puromycin protein synthesis inhibitor BumetanideNa⁺K⁺Cl⁻cotransport inhibitor Methyladenine [3-methyladenine] autophagicinhibitor H9 kinase inhibitor TPEN cell permeable heavy metal chelatorspermine NONOate NO donor

In addition to the compounds above, 5,056 compounds from ICCB'scommercial diversity set were screened, resulting in the identificationof PD00600, 5323069, and 1M556S as potential positives. A number of thepositives identified above, including alsterpaullone, H-9, curcumin,geldanamycin, AG370, Go6976, Spermine NONOate, PD00600, Nifedipine,α-amanitin, K252c, 5323069, 1M566S were further tested for their abilityto inhibit monoubiquitination of the FANC D2 polypeptide, and/or rendercisplatin hypersensitivity to 2008 cells expressing functional FANC F.The following examples describe the effects of three compounds, H-9,curcumin and alsterpaullone, in inhibiting the FA pathway.

Example 3 Identification and Characterization of H-9 as an Inhibitor ofFANC D2 Ubiquitination and Foci Formation

H-9 was identified as an inhibitor of the FA pathway using thehigh-throughput screen as described above. As shown by fluorescencemicroscopy in FIG. 7, H-9 inhibited the formation of FANC D2 foci in the50-100 μM range. As a secondary screen using immunoblot analysis todetermine the relative pools of monoubiquitinated FANC D2, H-9 treatmentwas found to decrease the overall level of FANC D2 monoubiquitination(FIG. 8). H-9 did not affect ATM-dependent phosphorylation of FANC D2but did inhibit ATR dependent phosphorylation of CHK1. Finally, thehypersensitivity of the FANC F deficient 2008 cells, which is restoredto wild-type levels when transfected with the FANCF cDNA, can bemimicked in 2008+FANCF cells by contacting the cells with curcumin (FIG.9). Taken together, these results suggest that H-9 blocks the FA/BRCApathway by inhibiting ATR kinase, either directly or indirectly.

Example 4 Identification and Characterization of Alsterpaullone as anInhibitor of FANC D2 Ubiquitination and Foci Formation

Likewise, alsterpaullone was identified as a potential inhibitor of theFA pathway using the high-throughput screen. Alsterpaullone is known toinhibit Cdk1/cycline B, Gsk-3B, and Cdk5 (Sausville et al. (2000) Ann NY Acad. Sci. 910:207-221; Schultz et al. (1999) J Med. Chem.42:2909-2919). Alsterpaullone inhibited the formation of FANC D2 foci ata concentration of 10 μM (FIG. 10). Like H-9, alsterpaullone inhibitedFANC D2 monoubiquitination and inhibited ATR dependent phosphorylationof Chk1 (FIG. 11).

Example 5 Identification and Characterization of Curcumin as anInhibitor of FANC D2 Ubiquitination and Foci Formation

The natural compound, curcumin, also caused a dose-dependent decrease inFANC D2 foci formation in the screening assay (data not shown).Furthermore, in the 3-20 micromolar range, curcumin caused a dosedependent decrease in FANC D2 monoubiquitination in HeLa cells and incisplatin-exposed HeLa cells (FIG. 10). Curcumin also caused adose-dependent decrease in ATR-mediated Chk1 phosphorylation (FIG. 11),further suggesting that curcumin blocks an upstream event in the FA/BRCApathway.

We next tested the ability of curcumin to potentiate the activity ofcisplatin in cytotoxicity assays. Curcumin sensitized FANCF-corrected2008 cells to cisplatin, but had a lesser effect on thechemosensitization of the parental 2008 cells (FIG. 12). Curcuminsensitized the cells in a dose range of 3-20 μM. This concentrationrange correlated with the curcumin dose range required for inhibition ofthe FA/BRCA pathway. Similar results were observed when the cells werepretreated for 24 hours with curcumin before the addition of cisplatin.Taken together, these results indicate that curcumin synergizes withcisplatin in enhancing ovarian tumor cytoxicity and that this curcumineffect correlates with its inhibition of the FA/BRCA pathway.

Next, the synergistic effects of curcumin in breast cancer cell lineswere assessed in order to see if the cytotoxic effects of cisplatincould be improved in cisplatin resistant cell lines. Thus, curcumin wastested for its effects in sensitizing a breast cancer cell line, MCF7(ATCC) to cisplatin. These results were similar to those seen with theovarian cancer cell lines. Both cisplatin and carboplatin demonstratedincreased cytoxicity with the addition of curcumin (5-20 μM). Thesedoses also resulted in decreased FANC D2 monoubiquitination in this cellline, indicating inhibition of the FA/BRCA pathway (FIG. 12). Chk 1phosphorylation was inhibited in all of the cell lines; 2008, 2008+F andMCF7 in a curcumin dose-dependent manner (FIG. 12).

The chemosensitizing activity of curcumin was further tested for itsspecificity to cisplatin. The ovarian tumor cell line, SKOV3, issensitive to cisplatin and taxol (Yang and Page, (1995) Oncol Res.7:619-24). Curcumin synergized with cisplatin in the killing of thesecells. In contrast, curcumin had no effect on the dose dependent taxolcytotoxicity profile of these cells.

Example 6 Correction of Cisplatin Hypersensitivity in the Ovarian CancerCell Line 2008 by FANCF Complementation

To determine whether these inhibitors are cisplatin-sensitizers twoovarian tumor lines—the cisplatin-sensitive, FANCF-deficient parental2008 line and the 2008 line corrected with the FANCF cDNA. The parental2008 line is deficient in FANCF due to epigenetic silencing of the FANCFgene (Taniguchi et al., (2003) Nat Rev Cancer. 3:23-34). Alsterpaulloneenhanced the cytotoxicity of cisplatin in the FANCF cDNA correctedcells, but not in the parental 2008 cells, indicating that this kinaseinhibitor is a potential cisplatin sensitizer and that it works throughinhibition of the FA pathway (FIG. 11). Curcumin sensitizedFANCF-corrected 2008 cells to cisplatin, but had a lesser effect on thechemosensitization of the parental 2008 cells (FIG. 9, and Table 3).Curcumin sensitized the cells in a dose range of 3-20 micromolar,corresponding to the curcumin dose range required for inhibition of theFA/BRCA pathway. Similar results were observed when the cells werepretreated for 24 hours with curcumin before the addition of cisplatin(data not shown). Taken together, these results indicate that curcuminsynergizes with cisplatin in enhancing ovarian tumor cytoxicity and thatthis curcumin effect correlates with its inhibition of the FA/BRCApathway.

Example 7 Determination of the Maximum Tolerated Dose forIntraventricular Administration of FA Pathway Inhibitors

FA pathway inhibitors Curcumin and Alsterpaullone are soluble at up to2.5 mM in 25% DMSO, the maximum concentration compatible withintraventricular administration. Taking into consideration that theentire mouse ventricular system consists of no more than 20 μl, themaximum concentration achieved (50 μM) exceeds that required for FApathway inhibition. Likewise, both AMD3100 and O6 Benzylguanine aresufficiently soluble in media, allowing estimated intraventricularconcentrations to exceed those required for efficacy in vitro. For eachcompound tested, a group of six mice are implanted with intraventricularcatheters (4 mm anterior to the lambda suture, 0.7 mm lateral ofmidline, and 2.5 mm below the dura). The catheters are connected to asubcutaneous Alzet Osmotic pump model 1007D (90 μl volume delivered at0.5 μl/hr) containing 2.5 mM, 1.25 mM, 0.625 mM, 0.313 mM, 0.151 mM ofeach compound or the vehicle media. The mice are monitored daily duringthe infusion period for neurologic deficits and sacrificed atpost-operative day 21. Brain sections are performed to 1) confirmcatheter continuity with the lateral ventricle, and 2) assess cellulartoxicity by stained with hematoxylin and eosin as well as TUNELstaining. The highest dose of drug delivered without clinical orhistologic evidence of damage are selected for subsequent efficacystudies, as detailed below.

Example 8 In vivo Tumoricidal Effects of Systemic BCNU Administration byIntravesicular Administration of an FA Pathway Inhibitor

The mouse xenograft, bioluminescence model used herein employs the U87GMB cell line (ATCC), which has been retrovirally transfected with thecoding sequence for luciferase in a pMMP vector. EstablishedU87-Luciferase cell lines are harvested in mid-logarithmic growth phase,resuspended as 50,000 cells in 10 μl PBS, and introduced into mice brainusing stereotactic guidance (2 mm lateral and posterior to the bregma, 3mm below dura). Mice are given D-luciferin (Xenogen, Alameda, Calif.)intraperitoneal injections and imaged with the IVIS imaging system(Xenogen) on post-surgery day 5 and 10.

For each compound tested, a group of 30 mice are implanted withU87-Luciferase and surveyed on post-implant day 5 and 10, out of which20-26 mice are expected to have uptake of the implanted tumor (Rubin etal., (2003) Proc. Natl. Acad. Sci. USA, 100: 13513-13518). Mice withU87-Luciferase tumor uptake are then surgically implanted with anintraventricular catheter and Alzet pump 1007D. These mice are thenstratified into four groups:

-   -   Group 1: treated with control vehicle (25% DMSO)    -   Group 2: treated with i.p. BCNU administration (15 mg/kg);    -   Group 3: treated with i.p. administration of an FA inhibitor at        the maximum tolerated dose; and    -   Group 4: treated with i.p. BCNU administration (15 mg/kg) as        well as FA inhibitor at the maximum tolerated dose.

The intraventricular catheter is placed contra-lateral to the tumorimplant site to minimize the effect of tumor growth on stereotacticcoordinates. The i.p. BCNU injection is carried out 4 days afterintraventricular administration to allow for sensitization. The mice areimaged on day 15 and 20 after initial tumor implant. Comparison of tumorgrowth is determined using LIVING IMAGE software package (Xenogen).

Example 9 Efficacy of FA Inhibitor in Sensitizing Ovarian Tumors toAnti-neoplastic Agents in an Animal Model

A number of animal models for ovarian cancer are known in the art. Forexample, Connolly et al. ((2003) Cancer Research, 63, 1389-1397), whichis incorporated herein by reference, discloses methods of developingepithelial ovarian cancer in mice by chimeric expression of the SV40 Tagunder control of the MISIIR promoter. In another example (Liu et al.,(2004) Cancer Research 64, 1655-1663), which is also incorporated hereinby reference, disclose the introduction of human HRAS or KRAS oncogenesinto immortalized human ovarian surface epithelial cells, which forms.c. tumors after injection into immunocompromised mice. These micemodels provide useful means to test the efficacy of FA inhibitors insensitizing ovarian tumors to anti-neoplastic agents. 6 mice are usedper group. To test the efficacy of cisplatin, alone or in combinationwith the FA inhibitor alsterpaullone, the following groups were used:

-   -   Group 1: treated with control vehicle    -   Group 2: treated with cisplatin, 4 mg/kg;    -   Group 3: treated with alsterpaullone at 5 mg/kg    -   Group 4: treated with cisplatin, 4 mg/kg, and alsterpaullone, 5        mg/kg. Repeat the cycle after two days.

All treatments are started a week after tumor inoculation. Mice aretreated for 10 cycles in total, and sacrificed for tumor nodule countingtwo weeks (on day 50) after discontinuation of drug treatment. Uponsacrifice, antitumor activity in each group is evaluated by counting thenumber of tumor nodules in the peritoneal cavity, measuring the diameterof the tumors, measuring the volume of the ascites and qualitativelyobserving the color of the peritoneal wall as an indication of thedegree of tumor-induced vascularization. Toxicity is evaluated byqualitative observation of the general appearance and behavior of themice prior to sacrifice and by measuring their body weight at variousintervals during the course of the treatments.

It will be clear to those skilled in the art that the efficacy of otherFA inhibitors, such as curcumin, alsterpaullone and H-9, can be testedusing this procedure. Curcumin is known to be safe at high doses, withLD₅₀ of greater than 10,000 mg/kg. Curcumin can be administered orally,intraperitoneally or intravesicularly. In one example, i.p.administration of curcumin at 100 mg/kg-300 mg/kg is performed, eitheralone or in combination with an anti-neoplastic agent (e.g., cisplatin)is tested in mice, as described above. In another example,intravesicular administration of curcumin is performed at the highestpossible dose determined using the method outlined above.

Example 10 Efficacy of a Combination of an FA Inhibitor and a DNA DamageRepair Pathway Inhibitor in Treating Ovarian Tumor

The efficacy of a combination of an FA inhibitor and an inhibitor of aDNA damage repair pathway is tested essentially as described above inExample 6. Briefly, the efficacy of the FA inhibitor (for example,curcumin, H-9, or alsterpaullone) in treating ovarian tumor, is testedalone or in combination with a DNA damage repair pathway inhibitor. Inone example, the following groups are tested:

-   -   Group 1: treated with control vehicle    -   Group 2: treated with alsterpaullone at 5 mg/kg    -   Group 3: treated with methoxyamine at 2 mg/kg    -   Group 4: treated with alsterpaullone, 5 mg/kg, and methoxyamine,        2 mg/kg. Repeat the cycle after two days.        Progress is monitored as previously described.

Example 11 Combination of an FA Inhibitor and a DNA Damage RepairPathway Inhibitor in Sensitizing Ovarian Tumors to Anti-NeoplasticAgents

In this example, the ability of a combination of an FA pathway inhibitorsuch as curcumin, H-9 or alsterpaullone, and a DNA damage repair pathwayinhibitor such as methoxyamine, in sensitizing a tumor toanti-neoplastic agents is tested using an animal model, essentially asdescribed above. However, the dosage of anti-neoplastic agentadministered can be varied to determine whether sensitization results ina lower overall dosage of the antineoplastic agent necessary to treatthe tumor. The following groups of mice are tested:

-   -   Group 1: treated with control vehicle    -   Group 2: treated with cisplatin, 0 mg/kg; alsterpaullone, 5        mg/kg, and methoxyamine, 2 mg/kg    -   Group 3: treated with cisplatin, 1 mg/kg; alsterpaullone, 5        mg/kg, and methoxyamine, 2 mg/kg    -   Group 4: treated with cisplatin, 2 mg/kg; alsterpaullone, 5        mg/kg, and methoxyamine, 2 mg/kg    -   Group 5: treated with cisplatin, 4 mg/kg; alsterpaullone, 5        mg/kg, and methoxyamine, 2 mg/kg        Progress is monitored as previously described.

TABLE 3 Chemosensitization of the Ovarian tumor line, 2008 + FANCF toCisplatin. Inhibitor 2008 2008 + FANCF Solvent (DMSO) S R Wortmannin S SH-9 S S Alsterpaullone S S Curcumin S S S, Hypersensitive to Cisplatin;R, Resistant to Cisplatin

Example 12 Clinical Evaluation of Treatment of Recurrent MullerianMalignancies With Curcumin and Carboplatin

A Phase I open-label, dose-escalation safety study is conducted inpatients with recurrent carcinoma of mullerian origin, less than 12months from prior platinum-based chemotherapy. Curcumin is administeredorally the night before, immediately prior to, the night of, and themorning following intravenous administration of carboplatin AUC 5. Atreatment cycle is 28 days with carboplatin adminstration beginning onday 1 followed by a 28-day follow-up period. Decisions regarding doseescalation and Dose Limiting Toxicity determination are made at the endof the 4 week cycle. Patients who tolerate treatment without evidence ofdisease progression are eligible for additional cycles ofcurcumin/carboplatin treatment.

Initially three patients will be entered in the first dose level. Theinitial dose level will be carboplatinum AUC 5 and curcumin 900 mg. Ifnone has Dose Limiting Toxicity (DLT), then the next 3 patients get doselevel 2. If a DLT occurs at any dose level, three additional patientsare enrolled to that dose level. If two DTLs occur at that dose level,then it is declared above the Maximum Tolerated Dose (MTD) and the MTDis defined at the previous dose level. No intrapatient dose escalationsare made.

Study Agent Administration

The initial dose will be carboplatin AUC 5 infused over 60 minutes andcurcumin 900 mg taken orally. During the study period, only the curcumindose will be escalated while the carboplatin dose will remain constantbased on the patient's renal function. A cycle is defined as an intervalof 28 days and is comprised of one treatment of carboplatin on Day 1 ofthe cycle and one course of curcumin administered the day prior (Day0),immediately prior to carboplatin on Day 1, the night of Day 1, and themorning of Day 2 for a total of 4 doses during each cycle. The doses ofcurcumin will be escalated for additional cohorts of patients until theDLT and MTD are determined.

Dose Escalation Schedule

Dose Level Carboplatin Curcumin 1 AUC 5  900 mg 2 AUC 5 1800 mg 3 AUC 52700 mg 4 AUC 5 3600 mg 5 AUC 5 4500 mgCarboplatin Administration

Carboplatin is infused intravenously over 1-hour. The dose ofcarboplatin is calculated as follows, using the Calvert formula based oncreatinine clearance:Total dose (mg)=Target AUC (in mg/ml per min)×(Estimated GFR+25)The carboplatin dose is calculated in mg, not mg/m². The initial targetAUC for carboplatin treatment in this trial is AUC=5. Creatinineclearance (CRCL) can either be measured, or estimated using the Jelliffeformula. Jelliffe formula For females:

${GFR} = \frac{0.9 \times \left( {98 - {0.8\left( {{age}^{*} - 20} \right)}} \right)}{{Cr}\left( {{mg}\text{/}{dl}} \right)}$* Age rounded to the nearest decadeIndividual preferences for carboplatin antiemetic pre-medication arepermitted. Typical pre-mediations include, zofran, ativan and decadron.

Definition of Dose-Limiting Toxicity (DLT)

The determination of DLT for purposes of assessing dose escalation isdefined as follows using the NCI CTC version 3.0 criteria withconsideration of known and accepted toxicities of carboplatin.Toxicities reached without pre-medication are not considered DLT.

-   -   Any nausea, vomiting >Grade 3 with maximum anti-emetic        pre-medication.    -   All other drug-related non-hematologic toxicity >Grade 3    -   Hematologic Toxicity        -   Neutrophil count <500 cell/ul for >7 days        -   Any febrile neutropenia (defined as T>101° F.) with a            neutrophil count <500 cells/ul after curcumin/carboplatin            administration        -   Platelet count <10,000 cell/ul OR Grade 3 with evidence of            bleeding necessitating blood product or platelet            transfusion.        -   Hemoglobin >Grade 4 toxicity with erythropoietin            co-administration            Evaluation of Response

Patients with measurable disease will be assessed by standard criteria.Patients are reevaluated after every two cycles of carboplatin/curcumin.In addition to a baseline/screening scan, confirmatory scans areobtained 4 weeks following initial documentation of an objectiveresponse.

Definitions

Response and progression are evaluated in this study using the newinternational criteria proposed by the Response Evaluation Criteria inSolid Tumors (RECIST) Committee (JNCI 92(3):205-216, 2000). Changes inthe largest diameter of the tumor lesions are used in the RECISTcriteria. Lesions are either measurable or nonmeasurable using thecriteria listed below.

Guidelines for Evaluation of Measurable Disease:

At baseline, tumors lesions are categorized as follows:

-   -   (1) measurable—lesions that can be accurately measured in at        least one dimension as 20 mm with conventional techniques or as        10 mm with spiral CT. OR    -   (2) nonmeasurable—all other lesions

All measurements are recorded in metric notation. All baselineevaluations are performed as closely as possible to the beginning oftreatment and never more than 4 weeks before the beginning of treatment.Non-measurable disease includes the following: bone lesions,leptomeningeal disease, ascites, pleural/pericardial effusion, abdominalmasses that are not confirmed and followed by imaging techniques, andcystic lesions.

Specifications by Methods of Measurements

The same method of assessment and the same technique is used tocharacterize each identified and reported lesion at baseline and duringfollow-up. Imaging-based evaluation is preferred to evaluation byclinical examination when both methods have been used to assess theantitumor effect of treatment.

Clinical Examination

Clinically detected lesions are considered measurable when they aresuperficial (i.e. skin nodules and palpable lymph nodes). All skinlesions are documented with color photography, including a ruler toestimate the size of the lesion.

Chest X-Ray

Although lesions on chest x-ray are acceptable as measurable lesionswhen they are clearly defined, a CT is preferable.

Computed Tomography (CT) and Magnetic Resonance Imaging (MRI)

CT and MRI are the best available (and most reproducible) methods formeasuring target lesions selected for response assessment. ConventionalCT and MRI are performed with contiguous cuts of 10 mm or less in slicethickness.

Ultrasound

Ultrasound is not used to measure tumor lesions. Ultrasound can beconsidered a possible alternative to clinical measurements forsuperficial palpable lymph nodes and subcutaneous lesions.

Tumor Markers

Tumor markers alone are not used to assess response. However, if markersare initially above the upper limit, they must return to normal levelsfor a patient to be considered in complete clinical response when alltumor lesions have disappeared.

Cytology, Histology

These techniques are used to differentiate between partial responses(PR) and complete responses (CR) in rare cases where residual lesions intumor types can contain benign components.

The cytological confirmation of the neoplastic origin of any effusionthat appears or worsens during treatment when the measurable tumor hasmet criteria for response or stable disease is mandatory todifferentiate between response or stable disease and progressivedisease.

Tumor Response Evaluation

Documentation of “Target” and “Nontarget” Lesions

All measurable lesions up to a maximum of 5 lesions per organ and 10lesions in total, representative of all involved organs, are identifiedas target lesions and recorded and measured at baseline. Target lesionsare selected on the basis of their size (those with the longestdiameter) and their suitability for accurate repeated measurements(either by imaging techniques or clinically). A sum of the longestdiameter for all target lesions is calculated and reported as thebaseline sum longest diameter. The baseline sum of the longest diameteris used as the reference by which to characterize the objective tumorresponse.

All other lesions (or sites of disease) are identified as nontargetlesions and also are recorded at baseline. Measurements of these lesionsare not required, but the presence or absence of each should be notedthroughout follow-up.

Response Criteria

Evaluation of Target Lesions

The criteria have been adapted from the original WHO Handbook, takinginto account the measurement of the longest diameter only for all targetlesions: complete response—the disappearance of all target lesions;partial response—at least a 30% decrease in the sum of the longestdiameter of target lesions, taking as reference the baseline sum longestdiameter; progressive disease—at least a 20% increase in the sum of thelongest diameter of target lesions, taking as reference the smallest sumlongest diameter recorded since the treatment started or the appearanceof one or more new lesions; stable disease—neither sufficient shrinkageto qualify for partial response nor sufficient increase to qualify forprogressive disease, taking as reference the smallest sum longestdiameter since the treatment started.

Evaluation of Nontarget Lesions

The criteria used to determine the objective tumor response fornontarget lesions are: complete response—the disappearance of allnontarget lesions and normalization of tumor marker level; incompleteresponse/stable disease—the persistence of one or more nontargetlesion(s) and/or the maintenance of tumor marker level above the normallimits; and progressive disease—the appearance of one or more newlesions and/or unequivocal progression of existing nontarget lesions.

Target Nontarget New Overall lesions lesions lesions response CR CR NoCR CR Incomplete response/SD No PR PR Non-PD No PR SD Non-PD No SD PDAny Yes or no PD Any PD Yes or no PD Any Any Yes PD CR = completeresponse; PR = partial response; SD = stable disease; and PD =progressive disease.

1. A method comprising: (a) providing a subject sample from a subjectwith a neoplastic disorder wherein said subject sample has been exposedto a genotoxic anti-neoplastic agent; (b) determining the number of FANCD2-containing foci in said subject sample; and (c) comparing the numberin (b) with the number of FANC D2-containing foci in a normal tissuesample that has been exposed to said genotoxic anti-neoplastic agent. 2.The method of claim 1, wherein the number of FANC D2-contaning foci isdetermined using an antibody specific for monoubiquitinated FANC D2. 3.The method of claim 1, wherein said exposure is less than or equal to atherapeutically effective dose.
 4. The method of claim 1, wherein saidexposure is at 50% or less of the therapeutically effective dose.
 5. Themethod of claim 1, wherein said normal tissue sample is of the same typeas said subject sample with said disorder.
 6. The method of claim 1,wherein said subject sample is exposed to said genotoxic anti-neoplasticagent in vivo or in vitro.
 7. The method of any one of the precedingclaims, wherein said genotoxic anti-neoplastic agent is selected fromthe group consisting of 1,3-bis(2-chloroethyl) -1-nitrosourea, busulfan,carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide,dacarbazine, daunorubicin, doxorubicin, epirubicin, etoposide,idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine,melphalan, mitomycin C, mitoxantrone, oxaliplatin, temozolomide,topotecan, and ionizing radiation.